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Prospective Evaluation of the Validity of Exhaled Nitric Oxide for the Diagnosis of Asthma^*

^* From the Department of Respiratory Medicine, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Belgium.

Correspondence to: Lieven J. Dupont, MD, PhD, Division of Respiratory Medicine, University Hospital Gasthuisberg, 49 Herestraat, B-3000 Leuven, Belgium; e-mail: lieven.dupont{at}uz.kuleuven.ac.be

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Study objective: Exhaled nitric oxide (NO) levels are significantly elevated in patients with inflammatory airways disorders such as asthma, and the measurement of exhaled NO has been proposed as a noninvasive marker of airways inflammation. The aim of this study was to assess the accuracy of exhaled NO levels for the diagnosis of asthma.

Methods: Two hundred forty consecutive, nonsmoking, steroid-naive patients, who were referred to our outpatient clinic with symptoms suggestive of obstructive airways disease, were investigated. Asthma was diagnosed in 160 patients on the basis of the presence of significant airways reversibility (FEV₁ > 12% predicted) and/or airways hyperresponsiveness (provocative concentration of histamine causing a 20% fall in FEV₁ 8 mg/mL). Prior to lung function measurements, exhaled NO was measured during a single-breath exhalation, according to European Respiratory Society and American Thoracic Society guidelines.

Results: The measurement of exhaled NO in our study population showed, at a cutoff level of 16 parts per billion, a specificity for the diagnosis of asthma of 90% and a positive predictive value of > 90%.

Conclusions: These findings suggest that the simple and absolutely noninvasive measurement of exhaled NO can be used as an additional diagnostic tool for the screening of patients with a suspected diagnosis of asthma.

Key Words: asthma • breath tests • diagnostic technique • exhalation • nitric oxide • noninvasive diagnostic method • pulmonary function tests

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Asthma is a chronic inflammatory disorder characterized by the presence of inflammatory cells and the release of several inflammatory mediators in the airways.¹ Airway inflammation is thought to result in airway obstruction, bronchial hyperresponsiveness, and remodeling of the airways.² The diagnosis and monitoring of asthma is based on conventional measurements, such as symptom scores reported by the patient, measurements of airway obstruction by means of peak expiratory flow (PEF) rates or FEV₁, assessment of bronchodilator response, and bronchial challenge tests to assess bronchial hyperresponsiveness.³ However, self-reporting of symptoms is much dependent on perception of symptoms with the potential for both underperception and overperception.⁴ Self-monitoring of PEF rate requires cooperation in producing maximal expiration and performing the measurements daily for several weeks. Although airway inflammation may be reflected by the degree of airway obstruction, the relationship of pulmonary function test results with objective indexes of inflammation is not a simple one.⁵ Moreover, patients with mild asthma frequently have normal baseline values of FEV₁. Challenge testing to methacholine or histamine is a reliable diagnostic test for airway hyperresponsiveness, with positive results in nearly all individuals,⁶ but the relationship to the degree of inflammation is not unique.

As inflammation is a central feature of bronchial asthma, directly measuring airway inflammation may, in some cases, be more appropriate for the diagnosis and monitoring of asthma. Until recently, only invasive techniques such as bronchoscopy could directly sample bronchial tissue and fluids for the presence of inflammatory cells and mediators. Examination of induced sputum is relatively noninvasive and produces valuable information on airway inflammation. However, induction of sputum may produce temporary decrements in lung function, and the processing of the sample is time-consuming, expensive, and requires skilled technicians.⁷ These methods are therefore not applicable in routine clinical practice.

Recently, the measurement of exhaled nitric oxide (NO) has been proposed as a noninvasive, simple test to assess airway inflammation in asthma. NO levels are significantly increased in exhaled air of patients with inflammatory airways disorders such as asthma.^{8 9 10} Treatment with inhaled glucocorticosteroids reduced these high NO levels in asthmatics in a dose-dependent manner¹¹ ; the level of exhaled NO has been shown to correlate with the degree of airway hyperresponsiveness,⁹ and the number of eosinophils in induced sputum and exhaled NO levels.¹² This suggests that NO in exhaled air might be used as a marker of airways inflammation, with a potential role in the diagnosis of asthma.

However, the value of this exhaled NO test in the diagnosis of asthma has yet to be determined. The aim of our study was, therefore, to assess the validity and accuracy of the measurement of exhaled NO in the diagnosis of asthma in patients with symptoms suggestive of asthma. We also wanted to determine a cutoff value of the measurement of exhaled NO for the diagnosis of asthma.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Patients
Patients were recruited among adult patients with symptoms suggestive of obstructive airway disease (eg, cough, wheezing, episodic dyspnea), who were consecutively referred to the asthma outpatient clinic of the university hospital Gasthuisberg, Leuven, Belgium, for diagnostic evaluation. In these patients, the diagnosis of asthma was further investigated by means of a thorough clinical assessment, pulmonary function tests, histamine challenge tests, and other diagnostic tests, when indicated, to determine the cause of the respiratory symptoms.

Two hundred forty patients were recruited, from whom informed consent was obtained. The exclusion criteria were the use of steroids or any other anti-inflammatory medications, current smoking (within past 6 weeks), and any other significant medical condition known to the patient or the referring physician. Patients and subjects did not consume any alcohol-containing or caffeinated beverages in the 4 h before the test, nor did they receive inhaled short-acting ß₂-mimetics in the 8 h prior to the measurements.

Study Design
This was a prospective study comparing the value of exhaled NO to conventional diagnostic tools in patients with a possible diagnosis of asthma. In each patient, exhaled NO was measured prior to pulmonary function tests and histamine challenge by an operator who was blinded to the patient history and diagnosis. Spirometry was measured both before and 15 min after inhalation of salbutamol, 400 µg, according to American Thoracic Society guidelines.¹³ Histamine challenge tests were performed according to the method of Cockcroft et al.¹⁴ The provocative concentration of histamine causing a 20% fall in FEV₁ (PC₂₀) was calculated by linear interpolation.

The clinical diagnosis was made by an experienced respiratory physician who was unaware of the exhaled NO level, based on the history and the results of the pulmonary function and provocation tests and other tests when indicated. The patients were subdivided for further analysis into two subgroups: asthmatics and nonasthmatics. Criteria for diagnosis of asthma were reversibility to ß₂-agonist 12% of predicted FEV₁ and/or PC₂₀ 8 mg/mL in accordance with the current international guidelines.³ Nonasthmatics failed to meet both criteria and underwent a more extensive diagnostic workup of the underlying cause of their respiratory symptoms.

Measurement of Exhaled NO
Exhaled NO was measured by means of an Eco Physics CLD 700 AL MED chemiluminescence analyzer (Eco Physics; Dürnten, Switzerland) adapted for on-line recording, during a single-breath exhalation, according to the European Respiratory Society and American Thoracic Society guidelines,^{15 16} and as previously published.⁹ In brief, subjects performed a slow expiratory vital capacity maneuver against a fixed expiratory resistance in order to have a constant flow of 200 mL/s. Exhaled air was continuously sampled from the exhalation limb of the system, and NO as well as CO₂ were measured. The point at which the CO₂ level reached 90% of its maximum value was identified, and the NO concentration was then determined by averaging the data collected over the next 5 s following the point at which the CO₂ level reached 90% of its maximum value. Three reproducible recordings were made, and the highest of three readings was used for analysis.

Statistical Analysis
Two-by-two contingency tables of exhaled NO (above vs equal to or below the cutoff point) vs asthma diagnosis (yes or no) were prepared using different levels of NO as cutoff point. Pretest probability, specificity, sensitivity, positive and negative predictive values, as well as test accuracy for each of these two-by-two tables were calculated. Accuracy of diagnosis was defined as the proportion of all tests that correctly identifies the presence or absence of the disease.

A receiver operating characteristic (ROC) curve was plotted, which allowed a graphical representation of sensitivity and specificity in order to view the best cutoff level for diagnosis. Comparisons between the asthmatic and the nonasthmatic groups were done by means of the Mann-Whitney U test for unpaired data; a p value of < 0.05 being considered significant.

	Results

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Patient Characteristics
The patient characteristics of both study groups are shown in Table 1 . Group A consisted of 160 patients with a diagnosis of asthma. Group B consisted of 80 patients in whom a diagnosis of asthma could not be established due to the absence of variable airway obstruction and/or airway hyperresponsiveness. There was no significant difference in age, female/male ratio, FEV₁, FVC, or FEV₁/FVC between both groups. A review of the medical records of the patients in group B (n = 80) demonstrated that the following diagnoses were made: postnasal drip and chronic rhinitis (n = 29), idiopathic chronic cough (n = 15), chronic sinusitis (n = 5), gastroesophageal reflux (n = 7), chronic bronchitis (n = 12), hyperventilation syndrome (n = 6), angiotensin-converting enzyme inhibitor-induced cough (n = 2), inhalation trauma (n = 1), bronchiectasis (n = 2), and thyroiditis (n = 1). The pretest probability of having asthma in our study population was 160 of 240 patients (67%).

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 1.. Scatter plot of the measurement of exhaled NO (ppb) in the asthmatic group and the in nonasthmatic group (p < 0.001). Line represents mean values.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 2.. ROC curve for the measurement of exhaled NO in the diagnosis of asthma. Data labels feature different cutoff points of exhaled NO levels.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

In the present study, we have further elaborated this hypothesis by analyzing the role of the exhaled NO level as a diagnostic test for asthma in a prospective manner in a large representative sample of consecutive patients presenting to our clinic with a possible diagnosis of asthma, in comparison with a standard diagnostic workup including pulmonary function testing, provocation testing, or measurement of PEF. The measurement of exhaled NO in our study population was associated with acceptable values of specificity, sensitivity, and accuracy of diagnosis for the diagnosis of asthma, depending on the cutoff value of exhaled NO.

The second aim of our study was to determine a cutoff value of the measurement of exhaled NO for the diagnosis of asthma. The main objective in deciding on a cutoff point is to strike the proper balance between false-positive and false-negative results. At a cutoff level equal to the average in normal adults at our setting (> 10 ppb), the sensitivity of the exhaled NO measurement was high (> 90%), although the specificity was low (50%). A higher cutoff point (eg, NO > 15 ppb) may increase the specificity up to nearly maximal levels, at the cost of lower sensitivity, indicating more false-negative results. This trade-off between specificity and sensitivity is also evident from the ROC curve in our study, which demonstrates a broad curvature. The cutoff point NO > 13 ppb was associated with the highest combination of specificity (80%) and sensitivity (85%), thus resulting in the best test accuracy. This "best" cutoff point was slightly higher than the upper limit of the 95% confidence interval (at a NO level of 11 ppb) in normal control subjects in our setting. This is probably due to the fact that patients without asthma did have other conditions that were responsible for their symptoms and that could also be associated with a small increase of the exhaled NO level.

Our results are in agreement with the recently reported findings by Chatkin et al,¹⁷ who showed similar levels of sensitivity and specificity of exhaled NO for the diagnosis of asthma in a study group of patients with chronic cough. The study group, however, was rather small, and extrapolation of these results may also be limited as only patients with chronic cough were included. NO was found to provide additional information compared to symptom questionnaire and methacholine provocation testing as a diagnostic test for asthma in a population study in adolescents.¹⁹ However, this was a cross-sectional study, which was further limited by excluding neither smokers nor patients receiving inhaled steroids, conditions that have been shown to reduce exhaled NO levels.^{9 10 11 20}

Our results on the sensitivity and specificity of exhaled NO are also comparable to the results with other diagnostic tools used to establish the diagnosis of asthma. The inhalation challenge test with methacholine or histamine is a highly reliable test for airway hyperresponsiveness, with positive results in nearly all individuals with current symptomatic asthma. This test should probably be regarded as the test with the best test accuracy, with a specificity of 100% and a sensitivity of 85%.⁶ Postbronchodilator responses (increase of FEV₁ 12%) and PEF variation ( 20%) have also been used as diagnostic test. However, these tests are much less accurate for detecting asthma, with a reported sensitivity (6% for postbronchodilator response and 10% for PEF variation at maximal specificity levels),⁶ which was lower than the sensitivity of the measurement of exhaled NO in our study population.

An important drawback to the implementation of exhaled NO as a diagnostic test for asthma is the large number of confounding factors that might influence the exhaled NO level.¹⁰ For the purpose of our study, we did not include smokers nor patients who had already been treated with inhaled steroids. Active smoking results in a significant reduction of the exhaled NO levels, both in normal subjects as well as in patients with asthma.^{10 20} Administration of inhaled steroids also results in a significant reduction of the exhaled NO levels in patients with asthma.^{9 10 11} The absolute values of exhaled NO are also dependent on the expiratory flow rate.²¹ Therefore, standardization of measurement technique is extremely important to allow comparisons across different studies.¹⁵ Although the measurement of NO in the exhaled air is noninvasive and simple, the equipment used to measure exhaled NO is expensive. Widespread clinical use of exhaled NO as a diagnostic test will certainly be more practicable if technological advances result in the development of smaller and less expensive analyzers, which can then be used in an outpatient setting.

It was also obvious from our results that the difference in test accuracy was not very large between cutoff levels ranging from > 10 to > 15 ppb, again reflecting the significant overlap of exhaled NO levels between asthmatics and nonasthmatics. Based on these findings, one could criticize the use of a single cutoff level of exhaled NO as a stand-alone diagnostic criterion for asthma in a clinical setting. Perhaps it would be more sensible to adopt a dual strategy to discriminate between asthmatics and nonasthmatics using both a high and a low cutoff point of exhaled NO as screening tool. Patients in our study population with an exhaled NO level not exceeding 8 ppb exhibit a low chance of having asthma, with a false-negative rate not exceeding 5%. In these patients, one could argue that it would be more worthwhile to look for other diseases than asthma as the cause for the reported symptoms; however, patients with an exhaled NO level > 18 ppb are very likely to have asthma, with a false-positive rate of < 5%. In these patients, one could advocate omitting further diagnostic workup such as challenge testing. In patients with NO levels between 9 ppb and 18 ppb, however, additional diagnostic tests are still indicated to confirm the suspected diagnosis. So by using exhaled NO as a screening test and applying this strategy, a significant number of additional diagnostic investigations could be avoided, possibly resulting in an important reduction of health-care costs. In our study population, implementation of this strategy would reduce the number of provocation tests by more than half.

In conclusion, we have shown in this specific study population that exhaled NO might be considered as an additional diagnostic test for asthma, with acceptable levels of sensitivity and specificity. Although an elevation of exhaled NO is not specific for asthma, the measurement of exhaled NO can be used in discriminating asthma from other disease conditions in patients with symptoms suggestive of obstructive airway disease.

	Footnotes

 
Abbreviations: NO = nitric oxide; PC₂₀ = provocative concentration of histamine causing a 20% fall in FEV₁; PEF = peak expiratory flow; ppb = parts per billion; ROC = receiver operating characteristic

Support was provided by a grant from the medical foundation Mathilde Horlait-Dapsens.

Received for publication December 20, 2001. Accepted for publication August 26, 2002.

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TOP Abstract Introduction Materials and Methods Results Discussion References

 

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