Clinical Use of Noninvasive Measurements of Airway Inflammation in Steroid Reduction in Children
http://www.100md.com
美国呼吸和危急护理医学 2005年第5期
Departments of Respiratory Pediatrics and Health Services Research, Clinical Trials and Evaluation Unit, Royal Brompton Hospital
National Heart and Lung Institute Clinical Studies Unit, Department of Thoracic Medicine, Imperial College, London, United Kingdom
Department of Pediatric and Adolescent Medicine, Pulmonary and Infectious Diseases, Wilhelminenspital, Vienna, Austria
Department of Pediatric Cardiology and Pulmonology, Heinrich Heine University, De箂seldorf, Germany
ABSTRACT
The use of noninvasive methods of monitoring airway inflammation, such as exhaled nitric oxide (eNO) and induced sputum, has been shown to improve asthma monitoring and optimize treatment in adult patients with asthma. There is a lack of comparable data in children. Forty children with stable asthma eligible for inhaled steroid reduction were reviewed every 8 weeks, and their inhaled steroid dose halved if clinically indicated. eNO, sputum induction combined with bronchial hyperreactivity testing, and exhaled breath condensate collection were performed at each visit to predict success or failure of reduction of inhaled steroids. Thirty of 40 (75%) children tolerated at least one dose reduction, 12 of 40 (30%) were successfully weaned off, and in total, 15 of 40 (38%) children experienced loss of asthma control. Treatment reduction was successful in all children who had no eosinophils in induced sputum before the attempted reduction. Using multiple logistic regression, increased eNO (odds ratio, 6.3; confidence interval, 3.75eC10.58) and percentage of sputum eosinophils (odds ratio, 1.38; confidence interval, 1.06eC1.81) were significant predictors of failed reduction. These findings suggest that monitoring airway inflammation may be useful in optimizing treatment in children with asthma.
Key Words: asthma children exhaled nitric oxide sputum eosinophil counts
Asthma is characterized by variable degrees of airway obstruction, airway hyperresponsiveness, and chronic airway inflammation. Current guidelines emphasize that inhaled corticosteroid (ICS) treatment is the mainstay of asthma therapy because it targets the underlying airway inflammation. The possible side effects of ICS, particularly when administered in high dosages, are of particular concern (1). It is therefore essential to use the lowest possible dose of ICS compatible with good asthma control (2). Current guidelines for asthma management recommend that, after a period of stability, a reduction of ICS dose should be attempted. In clinical practice, the decision to try to reduce ICS dosage is based on reported symptoms and lung function (3). Reports of symptoms are often inaccurate (4); spirometry depends on cooperation and, even in symptomatic children, is often within the normal range (5). A number of noninvasive methods of assessing inflammation have been developed, such as the measurement of exhaled nitric oxide (eNO), examination of the cellular and soluble content of induced sputum (sometimes in combination with a test of bronchial hyperresponsiveness [BHR]), and to some extent the analysis of exhaled breath condensate (EBC). Most of these methods have been used in cross-sectional studies, and very few of these studies have attempted to assess their utility to guide treatment in the asthma clinic. In a randomized study in adults, a strategy involving the measurement of BHR, and increasing ICS dose if there was an asymptomatic deterioration in this parameter, reduced asthma exacerbations and improved indices of airway remodeling over a 2-year period, at the cost of increased use of ICS (6). Green and colleagues (7) used sputum eosinophils to monitor treatment and showed that a strategy to normalize this parameter, regardless symptom control, reduced asthma exacerbations without increasing the use of ICS (7). Another study compared the ability of eNO, sputum eosinophils, and BHR to predict a relapse in asthma after stopping ICS, and showed that a rise in eNO predicted loss of control (8). Although eNO has been suggested as a potentially useful clinical tool in the management of asthma (9, 10), information is lacking on its use to monitor treatment in children in clinical practice. Despite the availability of methods of assessing airway inflammation, they are not routinely used to guide treatment, and there are no prospective longitudinal studies in children with asthma who are treated with ICS. The aim of this study was to assess the additional value of noninvasive markers of airway inflammation as predictors of success or failure of ICS reduction. We aimed to determine whether in the setting of a pediatric asthma clinic, measuring airway inflammation was of practical value in the management of the individual child, in whom clinically a reduction in ICS dosage was believed appropriate. Some of the results of this study have been previously reported in the form of an abstract (11, 12).
METHODS
Subjects
Forty children (age, 6eC17 years) with asthma (1) diagnosed by a pediatric respiratory physician (13) and (2) stable for at least 2 months (bronchodilator use < 3 times/week in past 2 months) on a constant ICS dose were recruited from the pediatric outpatients clinic.
Study Design
This was a prospective and observational study. Children eligible for ICS reduction according to current guidelines (3) were asked to participate. A follow-up visit was scheduled 2 months later unless a loss of asthma control occurred, in which case children were encouraged to attend for an immediate appointment. Full details of the dose reduction schedule are given in the online supplement. At each visit, a respiratory pediatrician unaware of the results of these markers made treatment decisions on the basis of clinical assessment and spirometry. The study was approved by the Royal Brompton Hospital Ethics Committee, and caregivers provided informed consent; children gave age-appropriate assent.
Assessment of Inflammation
eNO.
eNO was measured using a chemoluminescence analyzer (NIOX; Aerocrine, Stockholm, Sweden) according to published European Respiratory Society/American Thoracic Society guidelines (14).
Sputum induction with combined BHR testing.
Baseline spirometry was performed (Compact Vitalograph Ltd., Buckingham, UK) using at least three reproducible forced expiratory maneuvers to measure FEV1 and FVC (15). Sputum induction was performed using the following equipment: a DeVilbiss 2000 ultrasonic nebulizer (Somerset, PA); a Hans Rudolph 2700, two-way noneCre-breathing valve (Hans Rudolph, Inc., Kansas City, KS); and inhalation of hypertonic (4.5%) saline, as described elsewhere (16). If FEV1 was more than 75% of predicted at baseline and the child had not used a short-acting 2-agonist within the last 6 hours, we performed the procedure without premedication, allowing concurrent assessment of bronchial reactivity.
Sputum processing.
Selected sputum plugs were processed as previously reported (17). Differential cell counts using Diff QuickeCstained cytospins were performed by an investigator blind to the clinical status of the patient.
EBC.
Exhaled breath was collected for 10 minutes using an Echoscreen condenser (Jaeger; VIASYS Healthcare, Hoechberg, Germany) and samples stored immediately at eC80° C. We measured nitrite/nitrate and cysteinyl leukotriene levels as previously described (18, 19).
Definition of Loss of Asthma Control
Loss of asthma control was defined as the use of bronchodilators more than five times per week (excluding the prophylactic use before exercise or with a viral cold). In such a case, ICS treatment was increased, and the child withdrawn from the study. A course of oral steroids precluded a further reduction of inhaled steroids.
Statistical Analysis
Predictors of exacerbation were sought using multiple logistic regression, with robust variance estimation adjusting for the clustering of repeat visits with failure as outcome. Details can be found in the online supplement. Sensitivity and specificity for the predictors were calculated, and receiver operator characteristic curves constructed. Correlations were measured using Spearman rank correlation. Results were expressed as means and SD or medians and interquartile ranges (IQR), as appropriate; a p value less than 0.05 was considered statistically significant. All data were analyzed using Stata 7 (StataCorp LP, College Station, TX) (20).
RESULTS
Steroid Reduction
Demographic data are given in Table 1, and trial profile in Figure 1. After the first ICS reduction, 30 of 40 (75%) children were stable for at least 2 months.
Fifteen children tolerated further reduction steps successfully. Ten of 40 (25%) children had an exacerbation after the first reduction, and 5 of 40 (13%) after the subsequent reductions; therefore, 15 of 40 children were unable to tolerate a reduction in ICS despite one being clinically indicated. For those remaining stable, the median starting dose before reduction was 400 e (range, 200eC500) ICS. Similarly, the median daily dose in those who subsequently suffered an exacerbation during ICS reduction was also 400 e (range, 200eC800). The median ICS dose immediately before exacerbation was 250 e (range, 100eC750). The median time to exacerbation was 40 days (range, 25eC71). Complete weaning off was successful in 12 of 40 (30%) children. There was no difference in atopic status between children who subsequently exacerbated and those who remained stable.
Induced Sputum, BHR testing, eNO, and EBC
Sputum induction was successful in 75% of children, 88% of whom agreed to repeat the procedure at the next clinic visit. All children who failed to produce a sputum sample at the first visit were also unable to produce a sufficient sample at the next visit. The median weight of sputum sample was 1.2 g (IQR, 0.6eC2.1). eNO measurements were possible in the majority (89%) of children; results of BHR testing were eligible for analysis on 52 occasions (67%) where children fulfilled all criteria for bronchial hyperreactivity testing; EBC collection was possible in all the children.
Exacerbations.
There was evidence of increased airway inflammation during an exacerbation. In five cases, loss of control was only reported retrospectively as not all children were able to attend the clinic during an exacerbation (see Figure E1 in the online supplement for FEV1 values). During an exacerbation, the percentage of sputum eosinophils was significantly increased compared with the preceding stable visit (15.5% [IQR, 3eC22] vs. 0.5% [IQR, 0eC2], p < 0.005, paired data for n = 6). Similarly, eNO values were increased during an exacerbation (53 ppb [IQR, 29eC82] vs. 16 ppb [IQR, 8eC35], p < 0.05, paired data for n = 10). In contrast, neither nitrite/nitrate levels nor cysteinyl leukotrienes, measured in 22 and 21 children, respectively, increased before exacerbation, and the levels in children with stable asthma did not differ from levels found before or during an exacerbation (see Figures E2 and E3 in the online supplement).
Predicting result of ICS reduction.
Group findings.
The longitudinal measurements of sputum eosinophils and eNO for individual children during stepwise steroid reduction can be seen in Figure 2. Table 2 shows the odds ratios for percentage of sputum eosinophils, BHR testing, and eNO calculated from the individual models, adjusted for clustering of repeat visits. The percentage of sputum eosinophils was a significant predictor for failed reduction (exacerbation) in the single model. A positive BHR, defined as a fall of FEV1 of more than 15% from baseline, and a raised eNO ( 22 ppb) were also significant predictors for failed steroid reduction. However, this effect was not found when eNO was analyzed as a continuous variable.
In the combined logistic regression model, we only used the binary variable of eNO (eNO 22 ppb) because it had shown a strong statistical significance in the single model. Sputum eosinophils and eNO were statistically significant predictors of exacerbation, whereas BHR lost statistical significance in the multiple logistic regression model (Table 3).
Statistically significant correlations using Spearman rank correlations were found for eNO and sputum eosinophils, as well as for BHR and eosinophils and eNO (see Table E1 in the online supplement).
Data for prediction in individuals.
Sensitivity, specificity, and positive and negative predictive values of the inflammatory markers are shown in Table 4. The negative predictive value of BHR, eosinophils, or eNO was high, predicting a favorable outcome of treatment reduction in 84.6, 78.7, and 92.5%, respectively. The positive predictive value characterizing the percentage of children who subsequently experienced a loss of control with a positive test was 31.8% for a positive BHR test, 46.1% for sputum eosinophils, and 44% for an eNO of 22 ppb or greater.
Inhaled steroid reduction was successful in all children in the absence of eosinophils in sputum. Figure 3 shows the receiver operator characteristic curves for sputum eosinophils and eNO at various cutoff points.
DISCUSSION
This is the first article to report prospectively the predictive value of monitoring airway inflammation using noninvasive methods during ICS reduction in children with asthma. The percentage of sputum eosinophil and an eNO of 22 ppb or greater were significant predictors for failed ICS reduction in children with apparently well-controlled asthma. Using these methods as part of a clinical assessment of the individual patient could help optimize therapy because 22% (15 children in 71 visits) of children failed reduction, despite fulfilling clinical guideline criteria.
A retrospective analysis of the visits before exacerbation showed that excluding children with an eNO of 22 ppb or greater could have prevented an exacerbation in 11 of 14 (78%) children and, in the case of sputum eosinophils of 3% or more, an exacerbation could have been predicted in 8 of 11 (72%) children. On the other hand, if these values were used as indicators not to reduce treatment, a sputum eosinophil count of 3% or more would have prevented an attempted inhaled steroid reduction in 6 of 28 (21%) and elevated eNO levels in 19 of 49 (39%) occasions, where ICS reduction was in fact successful. Inhaled steroid reduction was always successful in asymptomatic children without sputum eosinophils.
This study was not designed to determine whether it is better to risk an exacerbation in some children by aggressively reducing ICS dose or "play for safety" at a risk of overtreatment. Clearly, however, this is an important question.
We found that above-normal eNO ( 22 ppb) was a significant predictor of failed ICS reduction. The odds ratio of more than 6 is high; however, this is a result from the group data analysis. This result seems less convincing when applied to the individual child. For example, using the same cutoff of eNO greater than 22 ppb as in the multiple logistic regression means that more than 20% of the children with eNO levels below this cutoff point will nevertheless fail ICS reduction. Similarly, more than 30% of children will remain stable during ICS reduction despite having elevated eNO levels. Combining the results of the percentage of sputum eosinophils, positive BHR, and elevated eNO in our model, sputum eosinophils and eNO were both significant predictors for exacerbation, whereas BHR lost significance. A combination using all three suggested methods increased the sensitivity to 60% and the specificity to 89% (Table 4).
Our study has a number of limitations. First, although we recruited 40 children, our results are based on only 15 exacerbations. We were unable to perform a power calculation before this study because of the lack of relevant previous pediatric data. Many (5 of 15) of these exacerbations could not be verified by the investigator because of the distances some of the children had to travel. Either a detailed history was taken and treatment instructions then given over the phone or, alternatively, some exacerbations were treated by local doctors and only reported retrospectively.
However, the drop in FEV1, and rise in eNO and sputum eosinophils in the children we were able to study confirmed that at least these exacerbations were genuine, and implies that the ones we were unable to study, defined by the families on the same clinical criteria, were also likely to be genuine. We tried to reduce ICS in the same manner in all children. Interestingly, on five visits, the clinicians wished to reduce the dose, but the family was unwilling to risk a deterioration, despite the child meeting nationally agreed criteria for a treatment reduction. Likewise, on some visits, either the family or the pediatrician or both believed that a smaller reduction than directed by the protocol would be safer. Although this was subjective and not evidence-based, it is likely to reflect clinical practice in the real world.
We aimed to assess loss of asthma control likely to be caused by the decreased steroid dosage. Because the effect of ICS on mild virus-related symptoms in asthma is controversial, and is difficult to quantify, transitory deterioration of symptoms occurring during a clinically diagnosed viral cold and temporarily requiring a more frequent use of short-acting bronchodilators was excluded, and did not preclude a further dose reduction in inhaled steroids. A different basis for poor control of asthma and viral-related symptoms has been supported by others (21). This approach, which only applied to a few children, seems to have been justified, because three of five children successfully continued with ICS reduction despite virus-related symptoms.
We also did not ask the children to perform peak flow measurements. This was a deliberate decision, because they have been shown to be unreliable in children, with studies suggesting that more than 60% of the data were fabricated after 2 weeks of monitoring (22eC24).
We also have no information on adherence to treatment. Metered dose inhalers with inbuilt recorders would have been helpful, but again, are not used in routine clinical practice, where adherence usually has to be taken on trust. Information concerning symptom control was obtained retrospectively from parents and children at the next clinic visit; however, in a routine clinical setting, this is normal practice.
In this study, EBC measurements of nitrite/nitrate levels and cysteinyl leukotrienes proved disappointing clinical tools. The small volumes of condensate obtained meant that we had to make arbitrary choices of what to measure, and our results cannot be taken to suggest that EBC has no role in the monitoring of asthma. In particular, and in retrospect, measurement of EBC pH (25) or levels of oxidants and antioxidants (26) might have been worthwhile. Further studies are needed to determine whether components of EBC other than those measured in this study would be valuable in monitoring children with asthma.
Our results are in many ways similar to those of adult studies addressing the importance of sputum eosinophils in monitoring disease status or predicting future symptoms. In one study, a higher sputum eosinophil count at baseline was found in patients with subsequent exacerbations (27), and Leuppi and coworkers (28) found percentages of sputum eosinophils to be significantly greater before failure of reduction. In children, eosinophils in sputum were reported to be higher in children with asthma (29, 30), and the change in number was significantly associated with symptoms (31). What is novel in our study is the use of a panel of markers prospectively and the demonstration that some may be helpful in guiding therapy.
Our results do differ in some respects from adult studies (7, 8), in which it was suggested that eNO and sputum eosinophilia could be used interchangeably. Jones and coworkers (8) reported eNO and sputum to be equally useful in predicting exacerbation in adults; however, in their study, eNO measurements were made at weekly intervals, compared with every 8 weeks in our study. This, however, is not likely to be practical until a device for home monitoring of eNO becomes available.
As previously reported, sputum induction with combined hypertonic saline challenge has been found to be well tolerated, safe, and highly successful in children with asthma (15). A response to hypertonic saline in children is most strongly associated with current asthma and allergy symptoms (32). BHR, however, is not a direct marker of airway inflammation; it has been suggested to be a separate dimension in the description of chronic asthma (33). A considerable number of children had used bronchodilators before attending their clinical appointment; therefore, it was only possible to perform a test of BHR in a subgroup of children. A larger number might have shown a statistical significance in the multiple logistic regression model. However, there was a correlation between BHR and sputum eosinophils and eNO. Therefore, we suggest this test may be a surrogate marker for successful reduction in children in whom eNO measurements or sputum induction cannot be performed. However, a further prospective study is required to confirm this suggestion.
In conclusion, we have shown for the first time in children that the use of methods to monitor airway inflammation can help predict success or failure of ICS reduction. The number of exacerbations was relatively small, but inspection of the data suggests that our conclusion that eNO and sputum eosinophil measurements may be helpful is likely to be correct, although the absolute predictive values may be less reliable. These results need to be confirmed by other workers in larger numbers before they can be recommended for widespread clinical use. The data presented here can be used to inform the design of future studies. However, we have presented data, which for the first time provide proof of concept that the use of measurements of airway inflammation may be useful to guide therapy in individual children, and not merely be used to define mechanisms by looking at overlapping differences between groups.
Acknowledgments
The authors thank Dr. Louise Donnelly for her expertise in the measurement techniques of nitrite/nitrate levels in exhaled breath.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
REFERENCES
Skoner D. Update of growth effects of inhaled and intranasal corticosteroids. Curr Opin Allergy Clin Immunol 2002;2:7eC10.
Wilson JW, Robertson CF. Inhaled steroids—too much of a good thing Med J Aust 2002;177:288eC289.
The BTS/SIGN British guidelines on the management of asthma. Thorax 2003;58(Suppl 1):i1eCi84.
Roberts EM. Does your child have asthma Parent reports and medication use for pediatric asthma. Arch Pediatr Adolesc Med 2003;157:449eC455.
Davis SD. Neonatal and pediatric respiratory diagnostics. Respir Care 2003;48:367eC385.
Sont JK, Willems LNA, Bel EH, van Krieken JH, Vanderbroucke JP, Sterk PJ, and the AMPUL Study Group. Clinical control and histopathologic outcome of asthma when using airway hyperresponiveness as an additional guide to long term treatment. Am J Respir Crit Care Med 1999;159:1043eC1051.
Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw A, Pavord ID. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002;360:1715eC1721.
Jones SL, Knittelson J, Cowan JO, Flannery EM, Hancox RJ, McLachlan CR, Taylor DR. The predictive value of exhaled nitric oxide measurements in assessing changes in asthma control. Am J Respir Crit Care Med 2001;164:738eC743.
Artlich A, Busch T, Lewandowski K, Jonas S, Gortner L, Falke KJ. Childhood asthma: exhaled nitric oxide in relation to clinical symptoms. Eur Respir J 1999;13:1396eC1401.
Strunk RC, Szefler SJ, Phillips BR, Zeiger RS, Chinchilli VM, Larsen G, Hodgdon K, Morgan W, Sorkness CA, Lemanske RF Jr. Childhood Asthma Research and Education Network of the National Heart, Lung, and Blood Institute. Relationship of exhaled nitric oxide to clinical and inflammatory markers of persistent asthma in children. J Allergy Clin Immunol 2003;112:883eC892.
Zacharasiewicz A, Wilson N, Lex C, Li A, Hansel T, Bush A. What markers help predict success or failure of steroid reduction in children Am J Respir Crit Care Med 2003;167:A295.
Zacharasiewicz A, Wilson N, Lex C, Donnelly L, Kharitonov S A, Bush A. Prospective nitrite, nitrate and cys-leukotriene(cys-LTs) measurements in EBC (exhaled breath condensate) in asthmatic children and relation to outcome during inhaled steroid reduction . Eur Respir J 2003;22:534s.
Von Mutius E. Presentation of new GINA guidelines for paediatrics. The Global Initiative on Asthma. Clin Exp Allergy 2000;30(Suppl 1):6eC10.
Baraldi E, de Jongste JC, for the European Respiratory Society/American Thoracic Society Task Force. Measurements of exhaled nitric oxide in children, 2001. Eur Respir J 2002;20:223eC237.
American Thoracic Society. Standardization of spirometry: 1994 update. Am J Respir Crit Care Med 1995;152:1107eC1136.
Jones PD, Hankin R, Simpson J, Gibson PG, Henry RL. The tolerability, safety, and success of sputum induction and combined hypertonic saline challenge in children. Am J Respir Crit Care Med 2001;164:1146eC1149.
Louis R, Shute J, Goldring K, Perks B, Lau LC, Radermecker M, Djukanovic R. The effect of processing on inflammatory markers in induced sputum. Eur Respir J 1999;13:660eC667.
Balint B, Donelly LE, Hanazawa T, Kharitonov SA, Barnes PJ. Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke. Thorax 2001;56:456eC461.
Czoma Z, Kharitonov SA, Balint A, Bush A, Wilson NM, Barnes PJ. Increased leukotrienes in exhaled breath condensate in childhood asthma. Am J Respir Crit Care Med 2002;166:1345eC1349.
Stata reference manual, release 7. 2. 2001;H-P:227eC228
Reddel H, Ware S, Marks G, Salome C, Jenkins C, Woolcock A. Differences between asthma exacerbations and poor asthma control. Lancet 1999;353:364eC369.
Redline S, Wright EC, Kattan M, Kercsmar C, Weiss K. Short-term compliance with peak flow monitoring: results from a study of inner city children with asthma. Pediatr Pulmonol 1996;21:203eC210.
Gorelick MH, Stevens M, Schultz T, Scribano PV. Difficulty in obtaining peak expiratory flow measurements in children with acute asthma. Pediatr Emerg Care 2004;20:22eC26.
Uwyyed K, Springer C, Avital A, Bar-Yishay E, Godfrey S. Home recording of PEF in young asthmatics: does it contribute to management Eur Respir J 1996;9:872eC879.
Vaughan J, Ngamtrakulpanit L, Pajewski TN, Turner R, Nguyen TA, Smith A, Urban P, Hom S, Gaston B, Hunt J. Exhaled breath condensate pH is a robust and reproducible assay of airway acidity. Eur Respir J 2003;22:889eC894.
Corradi M, Folesani G, Andreoli R, Manini P, Bodini A, Piacentini G, Carraro S, Zanconato S, Baraldi E. Aldehydes and glutathione in exhaled breath condensate of children with asthma exacerbation. Am J Respir Crit Care Med 2001;167:395eC399.
Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med 2000;161:64eC72.
Leuppi JD, Salome C, Jenkins CR, Anderson SD, Xuan W, Marks GB, Koskela H, Brannan JD, Freed R, Andersson M, et al. Predictive markers of asthma exacerbation during stepwise dose reduction of inhaled corticosteroids. Am J Respir Crit Care Med 2001;163:406eC412.
Gibson PG. Use of induced sputum to examine airway inflammation in childhood asthma. J Allergy Clin Immunol 1998;102:s100eCs101.
Gibson PG. How to measure airway inflammation: induced sputum. Can Respir J 1998;5(Suppl A):22AeC26A.
Gibson PG, Simpson J, Hankin R, Powell H, Henry RL. Relationship between induced sputum eosinophils and clinical pattern of childhood asthma. Thorax 2003;58:116eC121.
Riedler J, Gamper A, Eder W, Oberfeld G. Prevalence of bronchial hyperresponsiveness to 4.5% saline and its relation to asthma and allergy symptoms in Austrian children. Eur Respir J 1998;11:355eC360.
Rosi E, Ronchi MC, Grazzini M, Duranti R, Scano G. Sputum analysis, bronchial hyperresponsiveness, and airway function in asthma: results of a factor analysis. J Allergy Clin Immunol 1999;103:232eC237.(Angela Zacharasiewicz, Ni)
National Heart and Lung Institute Clinical Studies Unit, Department of Thoracic Medicine, Imperial College, London, United Kingdom
Department of Pediatric and Adolescent Medicine, Pulmonary and Infectious Diseases, Wilhelminenspital, Vienna, Austria
Department of Pediatric Cardiology and Pulmonology, Heinrich Heine University, De箂seldorf, Germany
ABSTRACT
The use of noninvasive methods of monitoring airway inflammation, such as exhaled nitric oxide (eNO) and induced sputum, has been shown to improve asthma monitoring and optimize treatment in adult patients with asthma. There is a lack of comparable data in children. Forty children with stable asthma eligible for inhaled steroid reduction were reviewed every 8 weeks, and their inhaled steroid dose halved if clinically indicated. eNO, sputum induction combined with bronchial hyperreactivity testing, and exhaled breath condensate collection were performed at each visit to predict success or failure of reduction of inhaled steroids. Thirty of 40 (75%) children tolerated at least one dose reduction, 12 of 40 (30%) were successfully weaned off, and in total, 15 of 40 (38%) children experienced loss of asthma control. Treatment reduction was successful in all children who had no eosinophils in induced sputum before the attempted reduction. Using multiple logistic regression, increased eNO (odds ratio, 6.3; confidence interval, 3.75eC10.58) and percentage of sputum eosinophils (odds ratio, 1.38; confidence interval, 1.06eC1.81) were significant predictors of failed reduction. These findings suggest that monitoring airway inflammation may be useful in optimizing treatment in children with asthma.
Key Words: asthma children exhaled nitric oxide sputum eosinophil counts
Asthma is characterized by variable degrees of airway obstruction, airway hyperresponsiveness, and chronic airway inflammation. Current guidelines emphasize that inhaled corticosteroid (ICS) treatment is the mainstay of asthma therapy because it targets the underlying airway inflammation. The possible side effects of ICS, particularly when administered in high dosages, are of particular concern (1). It is therefore essential to use the lowest possible dose of ICS compatible with good asthma control (2). Current guidelines for asthma management recommend that, after a period of stability, a reduction of ICS dose should be attempted. In clinical practice, the decision to try to reduce ICS dosage is based on reported symptoms and lung function (3). Reports of symptoms are often inaccurate (4); spirometry depends on cooperation and, even in symptomatic children, is often within the normal range (5). A number of noninvasive methods of assessing inflammation have been developed, such as the measurement of exhaled nitric oxide (eNO), examination of the cellular and soluble content of induced sputum (sometimes in combination with a test of bronchial hyperresponsiveness [BHR]), and to some extent the analysis of exhaled breath condensate (EBC). Most of these methods have been used in cross-sectional studies, and very few of these studies have attempted to assess their utility to guide treatment in the asthma clinic. In a randomized study in adults, a strategy involving the measurement of BHR, and increasing ICS dose if there was an asymptomatic deterioration in this parameter, reduced asthma exacerbations and improved indices of airway remodeling over a 2-year period, at the cost of increased use of ICS (6). Green and colleagues (7) used sputum eosinophils to monitor treatment and showed that a strategy to normalize this parameter, regardless symptom control, reduced asthma exacerbations without increasing the use of ICS (7). Another study compared the ability of eNO, sputum eosinophils, and BHR to predict a relapse in asthma after stopping ICS, and showed that a rise in eNO predicted loss of control (8). Although eNO has been suggested as a potentially useful clinical tool in the management of asthma (9, 10), information is lacking on its use to monitor treatment in children in clinical practice. Despite the availability of methods of assessing airway inflammation, they are not routinely used to guide treatment, and there are no prospective longitudinal studies in children with asthma who are treated with ICS. The aim of this study was to assess the additional value of noninvasive markers of airway inflammation as predictors of success or failure of ICS reduction. We aimed to determine whether in the setting of a pediatric asthma clinic, measuring airway inflammation was of practical value in the management of the individual child, in whom clinically a reduction in ICS dosage was believed appropriate. Some of the results of this study have been previously reported in the form of an abstract (11, 12).
METHODS
Subjects
Forty children (age, 6eC17 years) with asthma (1) diagnosed by a pediatric respiratory physician (13) and (2) stable for at least 2 months (bronchodilator use < 3 times/week in past 2 months) on a constant ICS dose were recruited from the pediatric outpatients clinic.
Study Design
This was a prospective and observational study. Children eligible for ICS reduction according to current guidelines (3) were asked to participate. A follow-up visit was scheduled 2 months later unless a loss of asthma control occurred, in which case children were encouraged to attend for an immediate appointment. Full details of the dose reduction schedule are given in the online supplement. At each visit, a respiratory pediatrician unaware of the results of these markers made treatment decisions on the basis of clinical assessment and spirometry. The study was approved by the Royal Brompton Hospital Ethics Committee, and caregivers provided informed consent; children gave age-appropriate assent.
Assessment of Inflammation
eNO.
eNO was measured using a chemoluminescence analyzer (NIOX; Aerocrine, Stockholm, Sweden) according to published European Respiratory Society/American Thoracic Society guidelines (14).
Sputum induction with combined BHR testing.
Baseline spirometry was performed (Compact Vitalograph Ltd., Buckingham, UK) using at least three reproducible forced expiratory maneuvers to measure FEV1 and FVC (15). Sputum induction was performed using the following equipment: a DeVilbiss 2000 ultrasonic nebulizer (Somerset, PA); a Hans Rudolph 2700, two-way noneCre-breathing valve (Hans Rudolph, Inc., Kansas City, KS); and inhalation of hypertonic (4.5%) saline, as described elsewhere (16). If FEV1 was more than 75% of predicted at baseline and the child had not used a short-acting 2-agonist within the last 6 hours, we performed the procedure without premedication, allowing concurrent assessment of bronchial reactivity.
Sputum processing.
Selected sputum plugs were processed as previously reported (17). Differential cell counts using Diff QuickeCstained cytospins were performed by an investigator blind to the clinical status of the patient.
EBC.
Exhaled breath was collected for 10 minutes using an Echoscreen condenser (Jaeger; VIASYS Healthcare, Hoechberg, Germany) and samples stored immediately at eC80° C. We measured nitrite/nitrate and cysteinyl leukotriene levels as previously described (18, 19).
Definition of Loss of Asthma Control
Loss of asthma control was defined as the use of bronchodilators more than five times per week (excluding the prophylactic use before exercise or with a viral cold). In such a case, ICS treatment was increased, and the child withdrawn from the study. A course of oral steroids precluded a further reduction of inhaled steroids.
Statistical Analysis
Predictors of exacerbation were sought using multiple logistic regression, with robust variance estimation adjusting for the clustering of repeat visits with failure as outcome. Details can be found in the online supplement. Sensitivity and specificity for the predictors were calculated, and receiver operator characteristic curves constructed. Correlations were measured using Spearman rank correlation. Results were expressed as means and SD or medians and interquartile ranges (IQR), as appropriate; a p value less than 0.05 was considered statistically significant. All data were analyzed using Stata 7 (StataCorp LP, College Station, TX) (20).
RESULTS
Steroid Reduction
Demographic data are given in Table 1, and trial profile in Figure 1. After the first ICS reduction, 30 of 40 (75%) children were stable for at least 2 months.
Fifteen children tolerated further reduction steps successfully. Ten of 40 (25%) children had an exacerbation after the first reduction, and 5 of 40 (13%) after the subsequent reductions; therefore, 15 of 40 children were unable to tolerate a reduction in ICS despite one being clinically indicated. For those remaining stable, the median starting dose before reduction was 400 e (range, 200eC500) ICS. Similarly, the median daily dose in those who subsequently suffered an exacerbation during ICS reduction was also 400 e (range, 200eC800). The median ICS dose immediately before exacerbation was 250 e (range, 100eC750). The median time to exacerbation was 40 days (range, 25eC71). Complete weaning off was successful in 12 of 40 (30%) children. There was no difference in atopic status between children who subsequently exacerbated and those who remained stable.
Induced Sputum, BHR testing, eNO, and EBC
Sputum induction was successful in 75% of children, 88% of whom agreed to repeat the procedure at the next clinic visit. All children who failed to produce a sputum sample at the first visit were also unable to produce a sufficient sample at the next visit. The median weight of sputum sample was 1.2 g (IQR, 0.6eC2.1). eNO measurements were possible in the majority (89%) of children; results of BHR testing were eligible for analysis on 52 occasions (67%) where children fulfilled all criteria for bronchial hyperreactivity testing; EBC collection was possible in all the children.
Exacerbations.
There was evidence of increased airway inflammation during an exacerbation. In five cases, loss of control was only reported retrospectively as not all children were able to attend the clinic during an exacerbation (see Figure E1 in the online supplement for FEV1 values). During an exacerbation, the percentage of sputum eosinophils was significantly increased compared with the preceding stable visit (15.5% [IQR, 3eC22] vs. 0.5% [IQR, 0eC2], p < 0.005, paired data for n = 6). Similarly, eNO values were increased during an exacerbation (53 ppb [IQR, 29eC82] vs. 16 ppb [IQR, 8eC35], p < 0.05, paired data for n = 10). In contrast, neither nitrite/nitrate levels nor cysteinyl leukotrienes, measured in 22 and 21 children, respectively, increased before exacerbation, and the levels in children with stable asthma did not differ from levels found before or during an exacerbation (see Figures E2 and E3 in the online supplement).
Predicting result of ICS reduction.
Group findings.
The longitudinal measurements of sputum eosinophils and eNO for individual children during stepwise steroid reduction can be seen in Figure 2. Table 2 shows the odds ratios for percentage of sputum eosinophils, BHR testing, and eNO calculated from the individual models, adjusted for clustering of repeat visits. The percentage of sputum eosinophils was a significant predictor for failed reduction (exacerbation) in the single model. A positive BHR, defined as a fall of FEV1 of more than 15% from baseline, and a raised eNO ( 22 ppb) were also significant predictors for failed steroid reduction. However, this effect was not found when eNO was analyzed as a continuous variable.
In the combined logistic regression model, we only used the binary variable of eNO (eNO 22 ppb) because it had shown a strong statistical significance in the single model. Sputum eosinophils and eNO were statistically significant predictors of exacerbation, whereas BHR lost statistical significance in the multiple logistic regression model (Table 3).
Statistically significant correlations using Spearman rank correlations were found for eNO and sputum eosinophils, as well as for BHR and eosinophils and eNO (see Table E1 in the online supplement).
Data for prediction in individuals.
Sensitivity, specificity, and positive and negative predictive values of the inflammatory markers are shown in Table 4. The negative predictive value of BHR, eosinophils, or eNO was high, predicting a favorable outcome of treatment reduction in 84.6, 78.7, and 92.5%, respectively. The positive predictive value characterizing the percentage of children who subsequently experienced a loss of control with a positive test was 31.8% for a positive BHR test, 46.1% for sputum eosinophils, and 44% for an eNO of 22 ppb or greater.
Inhaled steroid reduction was successful in all children in the absence of eosinophils in sputum. Figure 3 shows the receiver operator characteristic curves for sputum eosinophils and eNO at various cutoff points.
DISCUSSION
This is the first article to report prospectively the predictive value of monitoring airway inflammation using noninvasive methods during ICS reduction in children with asthma. The percentage of sputum eosinophil and an eNO of 22 ppb or greater were significant predictors for failed ICS reduction in children with apparently well-controlled asthma. Using these methods as part of a clinical assessment of the individual patient could help optimize therapy because 22% (15 children in 71 visits) of children failed reduction, despite fulfilling clinical guideline criteria.
A retrospective analysis of the visits before exacerbation showed that excluding children with an eNO of 22 ppb or greater could have prevented an exacerbation in 11 of 14 (78%) children and, in the case of sputum eosinophils of 3% or more, an exacerbation could have been predicted in 8 of 11 (72%) children. On the other hand, if these values were used as indicators not to reduce treatment, a sputum eosinophil count of 3% or more would have prevented an attempted inhaled steroid reduction in 6 of 28 (21%) and elevated eNO levels in 19 of 49 (39%) occasions, where ICS reduction was in fact successful. Inhaled steroid reduction was always successful in asymptomatic children without sputum eosinophils.
This study was not designed to determine whether it is better to risk an exacerbation in some children by aggressively reducing ICS dose or "play for safety" at a risk of overtreatment. Clearly, however, this is an important question.
We found that above-normal eNO ( 22 ppb) was a significant predictor of failed ICS reduction. The odds ratio of more than 6 is high; however, this is a result from the group data analysis. This result seems less convincing when applied to the individual child. For example, using the same cutoff of eNO greater than 22 ppb as in the multiple logistic regression means that more than 20% of the children with eNO levels below this cutoff point will nevertheless fail ICS reduction. Similarly, more than 30% of children will remain stable during ICS reduction despite having elevated eNO levels. Combining the results of the percentage of sputum eosinophils, positive BHR, and elevated eNO in our model, sputum eosinophils and eNO were both significant predictors for exacerbation, whereas BHR lost significance. A combination using all three suggested methods increased the sensitivity to 60% and the specificity to 89% (Table 4).
Our study has a number of limitations. First, although we recruited 40 children, our results are based on only 15 exacerbations. We were unable to perform a power calculation before this study because of the lack of relevant previous pediatric data. Many (5 of 15) of these exacerbations could not be verified by the investigator because of the distances some of the children had to travel. Either a detailed history was taken and treatment instructions then given over the phone or, alternatively, some exacerbations were treated by local doctors and only reported retrospectively.
However, the drop in FEV1, and rise in eNO and sputum eosinophils in the children we were able to study confirmed that at least these exacerbations were genuine, and implies that the ones we were unable to study, defined by the families on the same clinical criteria, were also likely to be genuine. We tried to reduce ICS in the same manner in all children. Interestingly, on five visits, the clinicians wished to reduce the dose, but the family was unwilling to risk a deterioration, despite the child meeting nationally agreed criteria for a treatment reduction. Likewise, on some visits, either the family or the pediatrician or both believed that a smaller reduction than directed by the protocol would be safer. Although this was subjective and not evidence-based, it is likely to reflect clinical practice in the real world.
We aimed to assess loss of asthma control likely to be caused by the decreased steroid dosage. Because the effect of ICS on mild virus-related symptoms in asthma is controversial, and is difficult to quantify, transitory deterioration of symptoms occurring during a clinically diagnosed viral cold and temporarily requiring a more frequent use of short-acting bronchodilators was excluded, and did not preclude a further dose reduction in inhaled steroids. A different basis for poor control of asthma and viral-related symptoms has been supported by others (21). This approach, which only applied to a few children, seems to have been justified, because three of five children successfully continued with ICS reduction despite virus-related symptoms.
We also did not ask the children to perform peak flow measurements. This was a deliberate decision, because they have been shown to be unreliable in children, with studies suggesting that more than 60% of the data were fabricated after 2 weeks of monitoring (22eC24).
We also have no information on adherence to treatment. Metered dose inhalers with inbuilt recorders would have been helpful, but again, are not used in routine clinical practice, where adherence usually has to be taken on trust. Information concerning symptom control was obtained retrospectively from parents and children at the next clinic visit; however, in a routine clinical setting, this is normal practice.
In this study, EBC measurements of nitrite/nitrate levels and cysteinyl leukotrienes proved disappointing clinical tools. The small volumes of condensate obtained meant that we had to make arbitrary choices of what to measure, and our results cannot be taken to suggest that EBC has no role in the monitoring of asthma. In particular, and in retrospect, measurement of EBC pH (25) or levels of oxidants and antioxidants (26) might have been worthwhile. Further studies are needed to determine whether components of EBC other than those measured in this study would be valuable in monitoring children with asthma.
Our results are in many ways similar to those of adult studies addressing the importance of sputum eosinophils in monitoring disease status or predicting future symptoms. In one study, a higher sputum eosinophil count at baseline was found in patients with subsequent exacerbations (27), and Leuppi and coworkers (28) found percentages of sputum eosinophils to be significantly greater before failure of reduction. In children, eosinophils in sputum were reported to be higher in children with asthma (29, 30), and the change in number was significantly associated with symptoms (31). What is novel in our study is the use of a panel of markers prospectively and the demonstration that some may be helpful in guiding therapy.
Our results do differ in some respects from adult studies (7, 8), in which it was suggested that eNO and sputum eosinophilia could be used interchangeably. Jones and coworkers (8) reported eNO and sputum to be equally useful in predicting exacerbation in adults; however, in their study, eNO measurements were made at weekly intervals, compared with every 8 weeks in our study. This, however, is not likely to be practical until a device for home monitoring of eNO becomes available.
As previously reported, sputum induction with combined hypertonic saline challenge has been found to be well tolerated, safe, and highly successful in children with asthma (15). A response to hypertonic saline in children is most strongly associated with current asthma and allergy symptoms (32). BHR, however, is not a direct marker of airway inflammation; it has been suggested to be a separate dimension in the description of chronic asthma (33). A considerable number of children had used bronchodilators before attending their clinical appointment; therefore, it was only possible to perform a test of BHR in a subgroup of children. A larger number might have shown a statistical significance in the multiple logistic regression model. However, there was a correlation between BHR and sputum eosinophils and eNO. Therefore, we suggest this test may be a surrogate marker for successful reduction in children in whom eNO measurements or sputum induction cannot be performed. However, a further prospective study is required to confirm this suggestion.
In conclusion, we have shown for the first time in children that the use of methods to monitor airway inflammation can help predict success or failure of ICS reduction. The number of exacerbations was relatively small, but inspection of the data suggests that our conclusion that eNO and sputum eosinophil measurements may be helpful is likely to be correct, although the absolute predictive values may be less reliable. These results need to be confirmed by other workers in larger numbers before they can be recommended for widespread clinical use. The data presented here can be used to inform the design of future studies. However, we have presented data, which for the first time provide proof of concept that the use of measurements of airway inflammation may be useful to guide therapy in individual children, and not merely be used to define mechanisms by looking at overlapping differences between groups.
Acknowledgments
The authors thank Dr. Louise Donnelly for her expertise in the measurement techniques of nitrite/nitrate levels in exhaled breath.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
REFERENCES
Skoner D. Update of growth effects of inhaled and intranasal corticosteroids. Curr Opin Allergy Clin Immunol 2002;2:7eC10.
Wilson JW, Robertson CF. Inhaled steroids—too much of a good thing Med J Aust 2002;177:288eC289.
The BTS/SIGN British guidelines on the management of asthma. Thorax 2003;58(Suppl 1):i1eCi84.
Roberts EM. Does your child have asthma Parent reports and medication use for pediatric asthma. Arch Pediatr Adolesc Med 2003;157:449eC455.
Davis SD. Neonatal and pediatric respiratory diagnostics. Respir Care 2003;48:367eC385.
Sont JK, Willems LNA, Bel EH, van Krieken JH, Vanderbroucke JP, Sterk PJ, and the AMPUL Study Group. Clinical control and histopathologic outcome of asthma when using airway hyperresponiveness as an additional guide to long term treatment. Am J Respir Crit Care Med 1999;159:1043eC1051.
Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw A, Pavord ID. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002;360:1715eC1721.
Jones SL, Knittelson J, Cowan JO, Flannery EM, Hancox RJ, McLachlan CR, Taylor DR. The predictive value of exhaled nitric oxide measurements in assessing changes in asthma control. Am J Respir Crit Care Med 2001;164:738eC743.
Artlich A, Busch T, Lewandowski K, Jonas S, Gortner L, Falke KJ. Childhood asthma: exhaled nitric oxide in relation to clinical symptoms. Eur Respir J 1999;13:1396eC1401.
Strunk RC, Szefler SJ, Phillips BR, Zeiger RS, Chinchilli VM, Larsen G, Hodgdon K, Morgan W, Sorkness CA, Lemanske RF Jr. Childhood Asthma Research and Education Network of the National Heart, Lung, and Blood Institute. Relationship of exhaled nitric oxide to clinical and inflammatory markers of persistent asthma in children. J Allergy Clin Immunol 2003;112:883eC892.
Zacharasiewicz A, Wilson N, Lex C, Li A, Hansel T, Bush A. What markers help predict success or failure of steroid reduction in children Am J Respir Crit Care Med 2003;167:A295.
Zacharasiewicz A, Wilson N, Lex C, Donnelly L, Kharitonov S A, Bush A. Prospective nitrite, nitrate and cys-leukotriene(cys-LTs) measurements in EBC (exhaled breath condensate) in asthmatic children and relation to outcome during inhaled steroid reduction . Eur Respir J 2003;22:534s.
Von Mutius E. Presentation of new GINA guidelines for paediatrics. The Global Initiative on Asthma. Clin Exp Allergy 2000;30(Suppl 1):6eC10.
Baraldi E, de Jongste JC, for the European Respiratory Society/American Thoracic Society Task Force. Measurements of exhaled nitric oxide in children, 2001. Eur Respir J 2002;20:223eC237.
American Thoracic Society. Standardization of spirometry: 1994 update. Am J Respir Crit Care Med 1995;152:1107eC1136.
Jones PD, Hankin R, Simpson J, Gibson PG, Henry RL. The tolerability, safety, and success of sputum induction and combined hypertonic saline challenge in children. Am J Respir Crit Care Med 2001;164:1146eC1149.
Louis R, Shute J, Goldring K, Perks B, Lau LC, Radermecker M, Djukanovic R. The effect of processing on inflammatory markers in induced sputum. Eur Respir J 1999;13:660eC667.
Balint B, Donelly LE, Hanazawa T, Kharitonov SA, Barnes PJ. Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke. Thorax 2001;56:456eC461.
Czoma Z, Kharitonov SA, Balint A, Bush A, Wilson NM, Barnes PJ. Increased leukotrienes in exhaled breath condensate in childhood asthma. Am J Respir Crit Care Med 2002;166:1345eC1349.
Stata reference manual, release 7. 2. 2001;H-P:227eC228
Reddel H, Ware S, Marks G, Salome C, Jenkins C, Woolcock A. Differences between asthma exacerbations and poor asthma control. Lancet 1999;353:364eC369.
Redline S, Wright EC, Kattan M, Kercsmar C, Weiss K. Short-term compliance with peak flow monitoring: results from a study of inner city children with asthma. Pediatr Pulmonol 1996;21:203eC210.
Gorelick MH, Stevens M, Schultz T, Scribano PV. Difficulty in obtaining peak expiratory flow measurements in children with acute asthma. Pediatr Emerg Care 2004;20:22eC26.
Uwyyed K, Springer C, Avital A, Bar-Yishay E, Godfrey S. Home recording of PEF in young asthmatics: does it contribute to management Eur Respir J 1996;9:872eC879.
Vaughan J, Ngamtrakulpanit L, Pajewski TN, Turner R, Nguyen TA, Smith A, Urban P, Hom S, Gaston B, Hunt J. Exhaled breath condensate pH is a robust and reproducible assay of airway acidity. Eur Respir J 2003;22:889eC894.
Corradi M, Folesani G, Andreoli R, Manini P, Bodini A, Piacentini G, Carraro S, Zanconato S, Baraldi E. Aldehydes and glutathione in exhaled breath condensate of children with asthma exacerbation. Am J Respir Crit Care Med 2001;167:395eC399.
Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med 2000;161:64eC72.
Leuppi JD, Salome C, Jenkins CR, Anderson SD, Xuan W, Marks GB, Koskela H, Brannan JD, Freed R, Andersson M, et al. Predictive markers of asthma exacerbation during stepwise dose reduction of inhaled corticosteroids. Am J Respir Crit Care Med 2001;163:406eC412.
Gibson PG. Use of induced sputum to examine airway inflammation in childhood asthma. J Allergy Clin Immunol 1998;102:s100eCs101.
Gibson PG. How to measure airway inflammation: induced sputum. Can Respir J 1998;5(Suppl A):22AeC26A.
Gibson PG, Simpson J, Hankin R, Powell H, Henry RL. Relationship between induced sputum eosinophils and clinical pattern of childhood asthma. Thorax 2003;58:116eC121.
Riedler J, Gamper A, Eder W, Oberfeld G. Prevalence of bronchial hyperresponsiveness to 4.5% saline and its relation to asthma and allergy symptoms in Austrian children. Eur Respir J 1998;11:355eC360.
Rosi E, Ronchi MC, Grazzini M, Duranti R, Scano G. Sputum analysis, bronchial hyperresponsiveness, and airway function in asthma: results of a factor analysis. J Allergy Clin Immunol 1999;103:232eC237.(Angela Zacharasiewicz, Ni)