Bronchial provocation tests in clinical practice

ABSTRACT Bronchial hyperresponsiveness, which consists of an exaggerated response of the airways to bronchoconstrictor stimuli, is one of the main characteristics of asthma, presented in nearly all asthmatic patients. Bronchial hyperresponsiveness may also be present in other diseases, such as allergic rhinitis, chronic obstructive pulmonary disease, cystic fibrosis, heart failure and respiratory infection, and with some medications, such as β-blockers. Bronchial provocation tests (also known as bronchial challenges) are used to evaluate bronchial responsiveness. These tests have become increasingly used over the last 20 years, with the development and validation of accurate, safe and reproducible tests, and with the publication of well-detailed protocols. Several stimuli can be used in a bronchial challenge, and they are classified as direct and indirect stimuli. There are many indications for a bronchial challenge. In this review, we discuss the main differences between direct and indirect stimuli, and the use of bronchial challenges in clinical practice, especially for confirming diagnoses of asthma, exercise-induced bronchoconstriction and cough-variant asthma, and for use among elite-level athletes.


IntroductIon
Bronchial responsiveness is characterized by a change in airway caliber in response to bronchoconstrictor and/or bronchodilator stimuli.Bronchial hyperresponsiveness (BHR) is defined as increased responsiveness in comparison with an expected response.This increased response of the airways can be measured by inhalation of specific or non-specific stimuli, and it should not be expected to be observed in normal subjects.
2][3] In other words, BHR is detected by an abnormal response to a bronchoconstrictor stimulus.
Bronchial hyperresponsiveness is one of the main pathophysiological characteristics of asthma and is present in nearly all asthmatic patients, especially during symptomatic episodes. 4It can also be found in other diseases, such as allergic rhinitis, chronic obstructive pulmonary disease, cystic fibrosis and heart failure, or after a respiratory infection, and with some medications like β-blockers. 3,4Hyperresponsiveness can explain many clinical features of diseases, such as coughing, wheezing, chest tightness or dyspnea, which can occur after allergen  or occupational exposure, physical activity, respiratory infections or medications. 3,46][7][8] In this review, we discuss the main differences between direct and indirect stimuli, and the use of bronchial challenges in clinical practice, especially for confirming the diagnoses of asthma, exercise induced bronchoconstriction and cough-variant asthma, and for evaluating dyspnea among elitelevel athletes.Systematic reviews and practice guidelines were searched in the Cochrane Library, PubMed, Scopus, Embase, Lilacs, and Medline using the keywords Bronchial Provocation Tests and Bronchial Challenge.Additionally, we included some relevant original studies (Table 1).

Bronchial provocation tests
Generally, bronchial responsiveness is measured in the laboratory as the change in airway caliber occurring after inhalation of a bronchoconstrictor stimulus. 2These tests have become increasingly used over the last 20 years, with the development of accurate, safe and reproducible bronchial challenges, and with the publication of well-detailed protocols.Bronchial challenge tests are easily performed in adults and children more than seven years of age. 3,6,9onchial challenges are performed by the inhalation of a bronchoconstrictor stimulus followed by assessment of its response in terms of lung function, especially forced expiratory volume in one second (FEV 1 ).When a fall in FEV 1 is achieved, usually between 10 and 20% from the baseline measurement, the test is stopped and a concentration or dose is calculated and named provocative concentration (PC) or provocative dose (PD), respectively (Figure 1). 3,6,9,10e absolute contraindications for bronchial challenges are severe airflow limitation (FEV 1 < 50% predicted or < 1.0 l), heart attack or stroke within the last three months, uncontrolled hypertension, systolic BP > 200 mmHg, or diastolic BP > 100 mmHg, and known aortic aneurysm.The relative contraindications are moderate airflow limitation (FEV 1 < 60% predicted or < 1.5 l), inability to perform acceptable-quality spirometry, pregnancy, nursing mothers, current use of cholinesterase inhibitor medication (for myasthenia gravis) and epilepsy requiring medical treatment. 3,6,9,10edications known to influence bronchial responsiveness should be withheld before the test: oral inhaled short-acting (e.g.salbutamol, terbutaline or fenoterol), medium-acting (e.g.ipratropium or theophylline) and long-acting (e.g.formoterol, tiotropium or salmeterol) bronchodilators should be withheld for 8, 24, and 48 hours, respectively; leukotriene modifiers for 24 hours; cromolyn sodium for 8 hours; nedocromil for 48 hours; and, except for methacholine challenge, antihistamines are withheld for their duration of action.Coffee, tea, cola drinks and chocolate should not be consumed on the day of the test.
The intervals between exposures and tests should be taken into account.In order to avoid effects on the results, intervals should be respected, as follows: one to three weeks after environmental antigens, three to six weeks after respiratory infections, months after occupational sensitizers, one week after air pollutants and days to months after chemical irritants.The room needs to be efficiently ventilated, and staff with active asthma should not perform the test or be present in the room where it takes place. 3,6,9,10rect and indirect stimuli Several stimuli are in use for bronchial challenges, and each may constitute a piece of the jigsaw puzzle on the pathophysiology of BHR and respiratory diseases.11 The bronchial stimuli can be classified into direct and indirect stimuli, according to the main/dominant mechanism through which they cause the bronchoconstriction. 12 This classification highlights the heterogeneity of the airway response to the different stimuli, and helps in understanding the differences in sensitivity and specificity of the stimuli and the effect of treatment on hyperresponsiveness. 5,10,12 Therefore, the results from different bronchial challenges should be interpreted according to the stimulus used and the disease phase and treatment.
Direct stimuli cause bronchoconstriction acting on the effector cells, such as airway smooth muscle cells, bronchial vascular endothelial cells and mucus-producing cells. 5The main direct stimulus used in clinical practice is methacholine.Indirect stimuli cause bronchoconstriction through action on another cells, such as inflammatory cells, epithelial cells and/or neuronal cells, which interact secondarily with these effector cells. 5,11The main indirect stimuli used in clinical practice are hypertonic saline, mannitol, bradykinin, exercise and eucapnic voluntary hyperpnea.Some stimuli are able to cause bronchoconstriction through both direct and indirect action.Several stimuli used for bronchial challenge are listed in Chart 1.
Both direct and indirect stimuli can be used as complementary information in diagnosing respiratory diseases, such as asthma, cough and exercise-induced bronchoconstriction (EIB).Although there are differences in the airway response to the different stimuli, our experience shows that it is more important, in general practice, to get used to one bronchoconstrictor stimulus and protocol (thus knowing its advantages and limitations), than to use several stimuli only few times each (thus knowing several stimuli superficially).
The major difference between direct and indirect stimuli, in general practice, is the sensitivity and specificity of each stimulus in diagnosing respiratory diseases, especially asthma.Bronchial challenge with direct stimuli, such as histamine and methacholine, is extremely sensitive for diagnosing asthma patients.However, these stimuli lack specificity, both in differentiating asthma from normal and asthma from chronic airflow limitation. 4Therefore, while a positive challenge in a symptomatic patient, especially with direct stimuli, does not confirm asthma, a negative test has a highly negative predictive value and rules out asthma.
Indirect stimuli are used in bronchial challenges because of their higher specificity for identifying people with active asthma, and to evaluate treatments with anti-inflammatory drugs. 5,10,12e major advantage of indirect stimuli is their capacity to act on many different cells, so that a wide variety of substances contribute towards the airway narrowing (e.g., histamine, leukotrienes, prostaglandins and neuropeptides), which makes the response more similar to bronchoconstriction developed during daily activities. 10,13,14Since indirect stimuli have higher specificity, another advantage is the lack of false-positive tests; hence the interest in these stimuli in epidemiological studies on asthma prevalence. 10,15It has been suggested that indirect stimuli would better reflect the degree of airway inflammation than would direct stimuli, with greater usefulness for evaluating and monitoring the response to anti-inflammatory drugs. 5,16,17

the use of bronchial provocation tests in general practice
There are many indications for a bronchial challenge, such as to confirm the diagnosis of asthma and/or EIB; to investigate cough- Despite the numerous indications, in general practice, bronchial challenges are mostly used for confirming the diagnoses of asthma, EIB and cough-variant asthma, and for assessing elitelevel athletes, as will be discussed subsequently. 4,10

the use of bronchial provocation tests in diagnosing asthma
A correct diagnosis for asthma is essential for adequate therapy.
Asthma generally manifests with symptoms of episodic breathlessness, wheezing, coughing and chest tightness and is confirmed through airflow limitation and reversibility, as detected through lung function tests (spirometry or peak expiratory flow). 18Other tests, such as measurements of allergic status, airway responsiveness and inflammation, can be used in atypical presentations. 18ong patients with symptoms consistent with asthma who present normal lung function, an objective measurement such as a bronchial challenge, with direct or indirect stimuli, may help establish a diagnosis of asthma. 10,19,20In these patients, detection of BHR suggests the diagnosis of asthma.The level of BHR in the bronchial challenge can also be used.Although there is no threshold that differentiates asthma from other diseases that present BHR, the BHR of asthma patients is usually more severe.Therefore, there is a correlation between the degree of airway responsiveness and asthma symptoms, 21,22 e.g. a histamine or methacholine PC 20 greater than 8 (or 16) mg/ml rules out current asthma in most instances, whereas a PC 20 less than 1 mg/ml is almost diagnostic of current asthma.Values between 1 and 8 mg/ml are intermediate in this regard. 23Since bronchial challenges have high sensitivity and limited specificity, a negative result can be useful for ruling out a diagnosis of persistent asthma, but a positive result does not always confirm the diagnosis. 18,21,23If the diagnosis after the bronchial challenge results remains uncertain, other tests, such as prick test, IgE measurement or induced sputum can be used as measurements of allergic status and airway inflammation, and/or a therapeutic test could be initiated.
It is important to evaluate the patient's clinical status and the medications used, given that the BHR is a dynamic process that can vary over time.It can increase after exposure to various environmental sensitizers and drugs (e.g.airborne allergens, substances found at the workplace, respiratory infections or propranolol), and it can decrease spontaneously or after anti-inflammatory therapy. 21,24,25

the use of bronchial provocation tests in diagnosing cough-variant asthma
Asthma is one of the most common etiologies among patients with a chronic cough.Other common diagnoses are postnasal drip syndrome and gastroesophageal reflux. 26,27Some patients with asthma have coughing as the sole symptom, and this is termed coughvariant asthma. 28Therefore, among patients with a chronic cough, asthma should always be considered as a potential etiology. 26ong patients with a chronic cough associated with reversible airway flow obstruction, the diagnosis of asthma should be considered and treatment for asthma should be initiated.However, among patients with a chronic cough and normal spirometry, additional tests should be done, including bronchial challenge.In these patients, the presence of BHR documented in a bronchial challenge suggests the diagnosis of asthma, and treatment should be initiated in order to confirm the diagnosis.Evaluation of treatment response is essential, since the diagnosis of asthma is confirmed only when a positive bronchial challenge is followed by a favorable response to asthma therapy, usually with the use of inhaled corticosteroids for one week. 29On the other hand, given the high specificity of bronchial challenges, a negative result rules out asthma from the differential diagnosis of chronic cough. 1 Methacholine has been the stimulus most frequently indicated and used in cough-variant asthma investigations.Some studies have demonstrated that indirect stimuli could also be used, but more studies are needed before that can be used in routine practice. 30,31

Bronchial provocation tests in exercise-induced bronchoconstriction
Exercise-induced bronchoconstriction (EIB) is characterized by an acute, transient airway narrowing that occurs during and most often after exercise. 32Common symptoms include coughing, wheezing, chest tightness and dyspnea, usually 5 to 10 min after exercise ceases, and they can remain significant for 30 min if no therapy is provided. 33EIB is more frequent after vigorous exercise and in cold and dry weather.EIB is found in 50 to 90% of all asthmatic patients and also occurs in up to 10% of subjects who are not known to be atopic or asthmatic. 34,35The prevalence of EIB among athletes is higher, and notifications by athletes have been increasing in recent Olympic Games. 32,36Because athletes without asthma may have been using inhaled β2-agonists in order to improve performance, it has been recommended that athletes should demonstrate current asthma, EIB or BHR in order to be approved to inhale β2-agonists at the Olympic Games. 36mong patients with a diagnosis of asthma, a complete history associated with therapeutic test is enough for a diagnosis of EIB.Among patients with symptoms of EIB and normal spirometry, and among elite athletes, bronchial challenges are necessary.
The most accurate tests for assessing EIB are exercise, eucapnic voluntary hyperpnea, hyperosmolar aerosols such as 4.5% saline, mannitol and methacholine. 32,36Because the airway response depends on the intensity of exertion and on environmental variables such as temperature, air humidity and time of the day, 37,38 it is crucial to know the conditions under which the bronchial challenge was done, in order to make an appropriate interpretation of its results. 39r elite athletes, the bronchial challenges accepted are exercise, eucapnic voluntary hyperpnea, hyperosmolar aerosols such as 4.5% saline, mannitol and methacholine.For exercise and eucapnic voluntary hyperpnea, a fall in FEV 1 of 10% is consistent with EIB; for hypertonic saline and mannitol, a fall in FEV 1 of 15% is considered to be BHR; and, for methacholine, a fall in FEV 1 of 20% confirms EIB diagnosis.Bronchial challenges with other stimuli, such as carbachol, histamine or adenosine monophosphate, have not been accepted by the International Olympic Committee's Medical Commission. 36Although laboratory-based challenges can be used for identifying EIB, field-based bronchial challenge has been found to be more sensitive. 40Moreover, the sensitivity of methacholine for identifying EIB in elite athletes has been reported to be low and less than 40%.Therefore, among elite athletes, a negative methacholine challenge does not completely rule out EIB, and an indirect stimulus can be used for further evaluation. 36,41,42Since many other conditions can mimic EIB, a detailed clinical history and physical examination should precede the bronchial challenge.

Perspectives
Bronchial challenges are useful in studying pathophysiological characteristics of respiratory diseases, such as the relationship between bronchial responsiveness and airway inflammation and remodeling.They could also be used as an index for asthma severity and clinical control.[45] Because of the lack of a reference diagnostic test (gold standard), it is difficult to determine the exact sensitivity and specificity of each different stimulus and for each disease.Some studies rely on physician diagnosis as the gold standard, but such assessments are highly subjective. 41veral studies have aimed to use bronchial challenge as an additional therapeutic choice.In cystic fibrosis, inhaled mannitol treatment significantly improved lung function. 46We have demonstrated that performing two hypertonic saline challenges in the evening attenuated the nocturnal fall in FEV 1 among asthmatic patients. 47idemiological studies on asthma have been hampered by lack of consistency in the results between reports.Most definitions of asthma have emphasized variable airflow obstruction and highlighted inflammation as essential elements of the condition.However, a positive BHR has been used as a relative standard criterion of validity.The association between positive BHR and symptoms has been seen as a gold standard definition of asthma and is a matter discussed in recent studies on asthma detection in large populations. 48,49Ferences

table 1 .Figure 1 .
Figure 1.Example of PD 20 calculation for hypertonic saline bronchial challenge obtained by interpolation between the last two points (second-to-last and final concentration) of the dose-response curve.

FEV 1 :
forced expiratory volume in one second PD 20 : provocative dose required to produce a 20% reduction in FEV 1 Bronchial challenges should be performed in the presence of staff with appropriate training for treating acute bronchoconstriction, including appropriate use of resuscitation equipment, which must be close enough to respond quickly to an emergency.
3,6,9,10chart 1. Direct and indirect stimuli used to measure bronchial responsiveness.Direct stimuli cause bronchoconstriction through action on effector cells and indirect stimuli cause bronchoconstriction through action on another cells, which interact secondarily with the effector cells.Some stimuli are able to cause bronchoconstriction through both direct and indirect action.The stimuli are classified according their main/dominant mechanism