INTRODUCTION

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Airway hyperresponsiveness (AHR) is considered a hallmark of asthma. However, the degree to which AHR predicts asthma severity remains enigmatic, particularly in children. Reports relating increased airway responsiveness to disease severity in children with asthma are inconsistent. In randomly selected populations of adolescents and young adults, increased airway responsiveness to methacholine is associated with more respiratory symptoms (1, 2), lower levels of lung function (2, 3), and increased morbidity (4). A number of primarily clinical studies relating airway responsiveness to frequent respiratory symptoms, levels of lung function, or other indices of disease severity have found no significant relationships (5). Some investigators have found methacholine challenge testing to be helpful in predicting disease severity in children (14, 15), whereas others have suggested a relationship to symptom score but not to level of lung function (16). The dearth of knowledge regarding the relationship between AHR and asthma severity in children is due in part to a lack of large cohort studies of children with asthma. In the context of the dramatic increase of asthma prevalence in early childhood, the ambiguous relationship between AHR and asthma severity in this age group merits further investigation. The present as study has as its aim to clarify the relationship between asthma severity and increased airway responsiveness in children with mild to moderate asthma using baseline data of 1,041 subjects enrolled in the Childhood Asthma Management Program (CAMP).

	METHODS

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Details of the design and methods of the CAMP trial are published elsewhere (17). Briefly, CAMP is a multicenter, randomized, double-masked clinical trial designed primarily to allow a comparison of the long-term safety and effectiveness of three inhaled treatments for mild to moderate childhood asthma: budesonide and albuterol, nedocromil and albuterol, and placebo and albuterol. A summary of the study population follows. CAMP enrolled 1,041 children ages 5 to 12 yr with mild to moderate chronic asthma over a 23-mo period. Entry criteria included asthma symptoms and/or medication use for 6 mo or more in the previous year. Eligibility for enrollment in the CAMP trial specified that children have chronic asthma as evidenced by one or more of the following historical findings for at least 6 mo in the year prior to interview: (1) asthma symptoms at least twice a week, (2) use of an inhaled bronchodilator at least twice a week, and (3) daily asthma medication. Study participants were excluded from enrollment in CAMP if they needed prednisone bursts more than five times over the preceding year, had more than one hospitalization for asthma in the year preceding their initial interview, or had ever required intubation for asthma. To further ensure that potential participants had current mild to moderate asthma, individuals were required to demonstrate current asthma of mild to moderate severity as manifested by a diary symptom score of  1 (scale 0-3) or A.M. or P.M. peak expiratory flow rate (PEFR) readings < 80% of personal best postbronchodilator value by diary on 8 or more days during a 28-d screening period while only on "as-needed" bronchodilator medication. Asthma was considered too severe for enrollment if, during the 28-d screening period, the child used more than eight puffs of albuterol for three consecutive days, had more than an average of 1.5 night awakenings due to asthma per week, had mean diary card symptom score greater than 2, or used medication other than albuterol to control asthma. All study participants evidenced at least a sensitivity to methacholine with provocative concentration of methacholine causing a 20% reduction in FEV₁ (PC₂₀)  12.5 mg/ml.

Study participants were excluded for the following reasons: FEV₁ < 65% of normal when off ₂-agonists for > 4 h and theophylline for > 24 h; other active pulmonary disease; pulmonary function suggestive of a restrictive ventilatory defect (i.e., symmetrically reduced FEV and FVC with normal ratios); evidence suggesting irreversible lung disease; severe chronic sinusitis or nasal polyposis; introduction of a change in allergy immunotherapy in the month before the interview; use of more than four sprays of nasal steroids daily (only beclomethasone allowed) at the time of randomization; current use of antigastroesophageal reflux medication; participation in another study; and pregnancy. Other exclusion criteria included the inability to perform acceptable spirometry, inability to complete the methacholine challenge test, and evidence that the patient's family might not adhere to study protocol requirements.

Data were collected by interview of parents/guardians by the research coordinator regarding demographics, history of asthma symptoms and severity, treatment of asthma, allergy history, characteristics of the home environment, and relevant family history. Each patient's parent or guardian signed a consent statement, and study participants signed an assent statement approved by each clinical center's institutional review board.

Pulmonary Function Testing

Spirometry and methacholine testing were performed by pulmonary function technicians trained and certified specifically for the CAMP protocol and procedures. Spirometry was performed on a Collins Stead-Wells dry-seal Survey III spirometer interfaced to a Dell desk-top personal computer and met or exceeded the American Thoracic Society (ATS) standards. At least three "acceptable" maneuvers had to demonstrate rapid onset of maximal flow, smoothness without hesitation or cough, continued exhalation until flow rate of zero, and a minimal expiration time of 6 s. In addition, at least two "reproducible" maneuvers were needed to show that FEV₁ and FVC were within 5% of best and PEFR was within 15% of best on no more than eight attempts. Spirometry and methacholine testing were performed at least 4 h after the use of a short-acting bronchodilator and 24 h after the last use of a long-acting bronchodilator. Postbronchodilator (two puffs albuterol by metered-dose inhaler) measurements were taken at each spirometry session (18). After administration of the bronchodilator, the minimal elapsed time before the postbronchodilator test was 15 min. Equations used to predict the average value of lung function measures for age, sex, and height were race-corrected according to Coultas and coworkers (19) for Hispanics, and according to Knudson and coworkers (20) for all other ethnic groups.

Airway responsiveness was determined by methacholine testing with the Wright nebulizer technique after the method of Cockcroft and coworkers modified for methacholine administration and by Juniper and coworkers (21, 22), and described in detail elsewhere (23) at least 4 h after the last use of a short-acting bronchodilator and at least 24 h after the last use of a long-acting bronchodilator. After a control diluent challenge, nine doubling doses of standardized methacholine (University of Iowa School of Pharmacy) ranging from concentrations of 0.098 to 25 mg/ml in 3 ml of solution were nebulized for 2 min each at 5-min intervals in a Wright nebulizer calibrated to a specific flowmeter (9 L/min) (Western Enterprises, Westlake, OH) to deliver a final output of 0.13 ml/min. Spirometry was performed 90 s after each challenge until FEV₁ had fallen by 20% or more. Methacholine sensitivity was expressed in terms of PC₂₀ by the following equation: PC₂₀ = antilog [log_e C1 + (log_e C2  log_e C1)(20  R1)/(R2  R1)], where C1 = second to last concentration tested, C2 = last concentration tested, R1 = percent fall in FEV₁ from diluent after C1, and R2 = percent fall in FEV₁ from diluent after C2. To minimize the effects of factors other than study medications and course of the asthma on methacholine reactivity, determinations of airway responsiveness were not made within 4 wk of an upper respiratory tract infection, other viral illness, or use of oral steroids or if the FEV₁ at baseline was less than 70% of predicted.

To provide standardization between clinical centers, methacholine solutions used in CAMP were prepared quarterly by the University of Iowa School of Pharmacy according to published methods (24). The solutions were first shipped to the drug distribution center for CAMP and then to the individual clinical centers. Periodically, methacholine solution stability was assayed by the Immunology Complement Laboratory at the National Jewish Medical and Research Center in Denver, Colorado.

Data Analysis

Data were analyzed at the Center for Clinical Trials of the Johns Hopkins School of Hygiene and Public Health. Baseline data collected for all study participants before randomization were analyzed. The distribution of PC₂₀ in the CAMP study participants at baseline was skewed, with a long right tail extending to 12.5 mg/ml, which corresponds to the least responsive study participants (Figure 1A). Because of this asymmetry, PC₂₀ was log transformed (Figure 1B) for all regression analyses. Data for four study participants who were responsive to diluent (i.e., 0.000 for their PC₂₀) were imputed with a value of 0.049, which is half the value of the lowest nonzero PC₂₀. Both unadjusted and adjusted regression analyses were performed for the following potential confounders: clinic, age, race, gender, education of the caregiver, income, season, time of day and calendar date of the methacholine challenge, familial asthma, and environmental tobacco smoke exposure.

View larger version (6K): [in this window] [in a new window]   View larger version (7K): [in this window] [in a new window]   Figure 1.   (A) Histogram of number of subjects by level of response to methacholine PC20. (B) Histogram of number of subjects by level of response to loge methacholine PC20.

Multiple linear regression (25) was used to test the association of PC₂₀ as a dependent variable with potential confounders of asthma severity and to test the association of PC₂₀ as an independent variable with measures of lung function, duration of asthma, and percentage of eosinophils as dependent variables. Multiple logistic regression (25) was also used to test the association of PC₂₀ as an independent variable with selected respiratory symptoms occurring at least weekly in the past 6 mo and clinical staff-assessed severity as dependent variables. For continuous outcomes, resistant regression techniques were used to minimize the effect of outliers by excluding observations with either large residuals or large influence defined as "far out" (26). Correlation coefficients were estimated with Spearman's rank correlation (22). p Values are two-sided and have not been adjusted for multiple comparisons or outcomes. All analyses were performed with SAS version 6.12 (27).

	RESULTS

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Table 1 presents descriptive data on the 1,038 childhood asthmatics participating in the CAMP study with nonmissing methacholine challenge data at baseline. All CAMP study participants had methacholine PC₂₀ less than or equal to 12.5 mg/ml because of eligibility constraints. The vast majority had a PC₂₀ less than 2 mg/ml (Figure 1A). Factors associated with methacholine PC₂₀ included clinic, date of testing, and family members' asthma status (Table 1).

Relationship of Methacholine Sensitivity to Level of Lung Function

Log_e methacholine PC₂₀ was related to prebronchodilator percent predicted FEV₁ (r = 0.29,  = 3.5, p < 0.0001) (Table 2). That is, for every log change in methacholine PC₂₀ there was a 3.5% predicted change in FEV₁ in liters. This relationship is such that the lower the PC₂₀ the lower is the level of FEV₁ percent predicted. Use of postbronchodilator FEV₁ values reduced the association of log_e methacholine PC₂₀ with FEV₁ percent predicted (r = 0.06,  = 0.30, p = 0.0005). Log_e methacholine PC₂₀ was most highly associated with FEV₁/FVC ratio and most weakly associated with FVC compared with FEV₁ (Table 2).

Relationship of Methacholine Sensitivity to Respiratory Symptoms

Table 3 presents the relationship of log_e methacholine PC₂₀ to the occurrence of chronic respiratory symptoms. A decreased methacholine PC₂₀ is associated with an increase in the number of symptomsshortness of breath, persistent wheezing, or exercise-induced cough and wheeze. The associations were strongest for the most frequently reported symptoms and weakest for nighttime symptoms (Table 3). During the screening period, before the methacholine challenge test, clinical staff assessed the severity of the child's asthma by answering the question: Would you classify this child's asthma as mild or moderate? (Table 3). There was a 22% increase in the adjusted relative odds of having moderate versus mild asthma per log decrease in methacholine PC₂₀ (p < 0.001) (Table 3). Log methacholine PC₂₀ was significantly and inversely related to peripheral blood eosinophil count (r = 0.36,  = 1.3, p < 0.0001, Table 2). For every 1.3% increase in peripheral blood eosinophil count there was a 1-log decrease in methacholine PC₂₀.

Because the National Asthma Education and Prevention Program (NAEPP) Guidelines advocate diary use to monitor peak flow, symptoms, and albuterol use as measures of asthma control, we assessed the relationship of information on these factors to methacholine PC₂₀ (Table 2). All diary measures were statistically significantly associated with methacholine PC₂₀, with the highest associations being obtained for albuterol use for symptoms, peak flow variability, and percentage of days with peak flow under 80% of personal best.

Duration of Asthma

Duration of asthma ranged from as short as 1 yr to as long as 12 yr among the CAMP subjects. PC₂₀ was significantly inversely related to asthma duration (r = 0.11,  = 0.05, p < 0.001) (Table 2, Figure 3), such that the lower the methacholine PC₂₀, the longer the duration of childhood asthma.

View larger version (20K): [in this window] [in a new window]   Figure 3.   PC20 versus duration of asthma.

	DISCUSSION

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The baseline asthma characteristics in a large cohort of children ages 5 to 12 yr with mild to moderate asthma have helped to clarify the relationship of methacholine airways responsiveness, expressed as log methacholine PC₂₀, to a variety of measures of asthma severity. Decreasing log methacholine PC₂₀ was found to be associated with lower levels of lung function (FEV₁ percent predicted), reports of asthma symptoms, subjective clinical staff assessment of asthma severity, longer duration of asthma, higher peripheral blood eosinophil count, and diary measures of asthma control. These results, owing to the precision derived from the large cohort size and reliability and consistency of spirometric and methacholine measurements, are to date the most consistent and unequivocal demonstration that methacholine PC₂₀ is modestly associated with multiple and diverse measures of disease severity in childhood asthma.

The inability of prior studies to yield definitive results on the relationship of airway responsiveness to asthma severity in children probably is due to their lack of statistical power and lack of assessment and control of confounding variables. These previous reports, generally evaluating fewer than 50 to 200 asthmatic participants, were too small to be able to detect the modest level of correlation observed in the CAMP data presented here (5). This problem is particularly acute in most clinical investigations of asthma study participants, including CAMP, because there is a tendency to truncate both the range of pulmonary function values and the range of methacholine airway responsiveness values in the clinical population under investigation, thus potentially reducing the opportunity to observe correlations. The CAMP selection criteria are clear and unequivocal and provide an objective definition of mild to moderate childhood asthma. Although the limited range of methacholine responsiveness may impair generalizability of our results to the very severe and very mild asthmatic children, the large sample size and clear severity criteria attest to the internal validity of our results. An increase in the asthma severity range might be expected to strengthen the observed association of AHR with asthma severity.

A number of studies have suggested a relationship between the concentration of inflammatory mediators and the degree of asthma severity (28). Baseline data in CAMP also demonstrate significant inverse associations between total blood eosinophil count and PC₂₀ (Table 2). The association of asthma duration with log_e methacholine PC₂₀ suggests that disease progression and chronic inflammation may be associated with worsening of airway responsiveness among asthmatic participants. The cross-sectional data presented here from the CAMP baseline database are inadequate to test this hypothesis; however, with the 5-yr longitudinal follow-up in CAMP, such relationships should be possible to determine. Moreover, CAMP's comparison of the long-term effects of anti-inflammatory treatment versus placebo on airway responsiveness should enable us to determine whether such treatment can modify the potential detrimental effects of chronic asthma.

The predictive power of clinicians to subjectively determine asthma severity and correlate it with degree of airway responsiveness is interesting but of unproven clinical utility. The level of overlap of log PC₂₀ is large and, although statistically significant, the means for the two groups (mild versus moderate asthma) are not distinct enough to have high predictive value.

The modest associations reported here do not justify an increase in the clinical application of methacholine challenge testing of asthmatic study participants to monitor response to therapy. Although there is clear consistency to these data with lung function, symptoms, physician assessment, and diary data, all being associated with level of methacholine airway responsiveness, the modest level of the correlation may not allow for high clinical predictability as a result of this test. This recommendation is controversial. European investigators are advocating the use of PC₂₀ as an index of adequacy of treatment and control in adults (32). This concept appears to await clinical confirmation, but the correlations with measures of ongoing symptoms and inflammation reported here are consistent with this approach. Because our data are cross-sectional, it will be important to test whether baseline methacholine will predict subsequent changes in disease severity and medication usage.

Our data are consistent with ongoing investigations demonstrating that chronic airway inflammation upregulates airway responsiveness (25). One of the central tenets of CAMP is intervention in this asthmatic process with anti-inflammatory medication. Whether such intervention effectively reverses or prevents asthma worsening or severity must await completion of this large trial in 1999.