Indirect airway challenges

Transcription

1 Eur Repir J 2003; 21: DOI: / Printed in UK all right reerved Copyright #ERS Journal Ltd 2003 European Repiratory Journal ISSN ERS TASK FORCE Indirect airway challenge G.F. Joo, B. O9Connor, on behalf of the Tak Force Co-author of the Tak Force Report: G.F. Joo (Chairman), B. O Connor (Co-Chairman), S.D. Anderon, F. Chung, D.W. Cockcroft, B. Dahlén, G. DiMaria, A. Forei, F.E. Hargreave, S.T. Holgate, M. Inman, J. Lötvall, H. Magnuen, R. Poloa, D.S. Potma, J. Riedler* Indirect airway challenge. G.F. Joo, B. O9Connor, on behalf of the Tak Force, Co-author of the Tak Force Report: G.F. Joo (Chairman), B. O Connor (Co-Chairman), S.D. Anderon, F. Chung, D.W. Cockcroft, B. Dahlén, G. DiMaria, A. Forei, F.E. Hargreave, S.T. Holgate, M. Inman, J. Lötvall, H. Magnuen, R. Poloa, D.S. Potma, J. Riedler. #ERS Journal Ltd ABSTRACT: Indirect challenge act by cauing the releae of endogenou mediator that caue the airway mooth mucle to contract. Thi i in contrat to the direct challenge where agonit uch a methacholine or hitamine caue airflow limitation predominantly via a direct effect on airway mooth mucle. Direct airway challenge have been ued widely and are well tandardied. They are highly enitive, but not pecific to athma and can be ued to exclude current athma in a clinic population. Indirect bronchial timuli, in particular exercie, hyperventilation, hypertonic aerool, a well a adenoine, may reflect more directly the ongoing airway inflammation and are therefore more pecific to identify active athma. They are increaingly ued to evaluate the prevalence of bronchial hyperreponivene and to ae pecific problem in patient with known athma, e.g. exercie-induced bronchocontriction, evaluation before cuba diving. Direct bronchial reponivene i only lowly and to a modet extent, influenced by repeated adminitration of inhaled teroid. Indirect challenge may reflect more cloely acute change in airway inflammation and a change in reponivene to an indirect timulu may be a clinically relevant marker to ae the clinical coure of athma. Moreover, ome of the indirect challenge, e.g. hypertonic aline and mannitol, can be combined with the aement of inflammatory cell by induction of putum. Eur Repir J 2003; 21: Correpondence: G.F. Joo Dept Repiratory Dieae Ghent Univerity Hopital De Pintelaan 185 B-9000 Ghent Belgium Fax: guy.joo@rug.ac.be Keyword: Adenoine athma bronchoprovocation chronic obtructive pulmonary dieae exercie methacholine Received: September Accepted: October CONTENTS Definition and main propertie of an indirect challenge Mechanim and receptor involved in indirect challenge Mechanim involved in the airway narrowing to phyical timuli: evidence from tudie on exercieinduced bronchocontriction Mechanim involved in the airway narrowing caued bypharmacological timuli: evidence on adenoine-, tachykinin-, and bradykinin-induced bronchocontriction Diagnotic value of the indirect challenge: a comparion with direct challenge Diagnotic value of the direct challenge, hitamine and methacholine Diagnotic value of indirect challenge Direct veru indirect airway challenge to monitor athma Ue of indirect airway challenge in epidemiological tudie Safety apect of indirect airway challenge Concluion Area for future reearch Mechanim and receptor Diagnoi Monitoring Epidemiology Safety, performance Appendix 1: Safety iue for exercie challenge in the lung function laboratory and in field tudie Appendix 2: Safety iue for airway challenge with hypertonic aline in the lung function laboratory and in field tudie Appendix 3: Performance tandard, afety iue and protocol recommendation for airway challenge with adenoine Appendix 4: Performance tandard, afety iue and protocol recommendation for airway challenge with lyine-apirin

2 INDIRECT AIRWAY CHALLENGES Bronchial hyperreponivene i an abnormal increae in airflow limitation following expoure to a nonallergic timulu [1, 2]. Bronchial hyperreponivene i a characteritic feature of both athma and chronic obtructive pulmonary dieae (COPD). Thu, bronchial hyperreponivene i frequently ued to aid in diagnoi and characteriation of individual with airway dieae. Although bronchial hyperreponivene i not pecific for athma, nearly all patient with athma exhibit increaed reponivene, which i more marked during ymptomatic epiode. Bronchial hyperreponivene to methacholine i alo preent in a majority of patient with mild to moderate COPD [3]. Moreover, the everity of bronchial hyperreponivene predict the repone to inhaled corticoteroid in patient with athma [4] and the progreion of airflow limitation in patient with COPD [5]. Mot invetigator ae bronchial reponivene uing methacholine or hitamine a a provocative timulu. Methacholine and hitamine caue airflow limitation predominantly via a direct effect on airway mooth mucle. By contrat, indirect challenge induce airflow limitation by acting on cell other than mooth mucle cell e.g. inflammatory cell, epithelial cell and nerve, which upon timulation releae mediator or neurotranmitter that provoke mooth mucle contraction. Nearly all the publihed tudie on athma and COPD have utilied hitamine and methacholine provocation tet for clinical characteriation of patient. Furthermore, hyperreponivene teting i widely ued in clinical reearch etting to evaluate potential new therapie. For example, direct challenge with hitamine or methacholine are ued to etablih a doe repone and time coure of the acute bronchoprotective effect of b-agonit. Thee challenge have alo been ued to ae the potential anti-inflammatory effect of prolonged treatment with new agent. There are limitation to thi model. Inhaled corticoteroid, the current gold tandard anti-inflammatory treatment for athma, reduce bronchial reponivene to hitamine or methacholine only to a mall degree, an effect that i both doe and time dependent. In recent year an increaing number of tudie have invetigated the relative uefulne of indirect airway challenge in monitoring anti-inflammatory treatment in athma, but almot none in COPD. In 1998 the European Repiratory Society (ERS) approved a Tak Force on Indirect Airway Challenge. The objective of thi Tak Force were to develop recommendation concerning the role of indirect airway challenge in the aement and monitoring of airway dieae. The recommendation in thi report are baed on a review of the publihed literature and were developed during workhop held at the American Thoracic Society in San Diego (April 1999), ERS Congre in Madrid (October 1999), ERS Meeting in Ghent (June 2000) and the ERS Congre in Florence (Augut 2000). The following topic were included. 1) Mechanim and receptor involved in the airway narrowing caued by indirect airway challenge. 2) Diagnotic value of indirect challenge. 3) Value of indirect challenge in the monitoring of athma, including the ue of thee challenge a an outcome meaure in clinical trial. 4) Value of indirect challenge in epidemiological tudie. 5) The importance of tandardiation of challenge method. 6) Area for further reearch. Definition and main propertie of an indirect challenge The concept of indirect challenge wa developed at the end of the eightie [6]. Several publication had confirmed that many different nonpecific timuli induced airway narrowing in patient with athma. Thu a ditinction had to be made between direct and indirect timuli. Methacholine and hitamine are direct timuli becaue they caue airflow limitation by acting on effector cell, predominantly on airway mooth mucle but alo on mucu gland and on airway microvaculature without involving intermediate pathway. By contrat indirect timuli, i.e. phyical timuli uch a exercie, omotic challenge or pharmacological timuli uch a adenoine, caue airflow limitation by acting on cell, mot notably inflammatory cell and neuronal cell which releae mediator or cytokine to caue econdary bronchocontriction. The fact that the pattern of airway narrowing induced by indirect timuli differ from that provoked by direct timuli i hown by the following clear evidence. 1) Bronchial reponivene to direct and indirect challenge are rather poorly correlated with each other [6]. 2) A wide array of mediator including hitamine, leukotriene, protaglandin, acetylcholine, neuropeptide are involved in the airway narrowing induced by the indirect timuli [7]. 3) The airway narrowing caued by an indirect, but not a direct challenge, can be prevented by acute pretreatment with a cromone (cromoglycate, nedocromil), inhaled fruemide and/or heparin [7]. 4) After the adminitration of an indirect challenge tachyphylaxi to a econd timulu, with the ame or another indirect acting agent (cro refractorine) i frequently oberved [7]. The tachyphylaxi oberved with the indirect challenge, i far more pronounced than the mall change een when hitamine or methacholine i repeatedly inhaled [8 10]. 4) In patient with athma, bronchial reponivene to an indirect airway challenge i more cloely aociated with airway inflammation than bronchial reponivene to a direct timulu [11]. Bronchial reponivene to an indirect timulu may alo better reflect acute change in airway inflammation induced by allergen avoidance [12] or by treatment with inhaled teroid [13, 14]. The author propoe the following practical, working definition of an indirect challenge: "Indirect challenge act by cauing the releae of endogenou mediator that caue the airway mooth mucle to contract, with or without effect in inducing microvacular leakage. Becaue the repone to thee challenge are modified or even completely inhibited by inhaled teroid, the airway repone to thee challenge may be a cloer reflection of active airway inflammation". Table 1. Overview of direct and indirect timuli Indirect timuli Direct timuli Phyical timuli Cholinergic agonit Exercie (acetylcholine, Noniotonic aerool methacholine, (hyper-, hypotonic, carbachol) ditilled water Hitamine aerool, mannitol) Protaglandin D 2 Eucapnic voluntary Leukotriene C 4 /D 4 /E 4 hyperpnoea of dry air Pharmacological timuli Adenoine Tachykinin Bradykinin Metabiulphite/SO 2 Propranolol Endotoxin (LPS) Platelet activating factor Ozone Selective agent Apirin and NSAID Allergen LPS: lipopolyacharide; NSAID: nonteroidal anti-inflammatory drug; SO 2 : ulphur dioxide.

3 1052 G.F. JOOS ET AL. Hyperpnoea Hypertonic Water lo Ditilled water Water gain Activated mat cell Eoinophil Senory nerve Leukotriene Hitamine NK 1 /NK 2 Allergen Leukotriene Hitamine Adenoine Hitamine AMP P Subtance P NKA Leukotriene NK 1 /NK 2 Bradykinin Apirin AA Leukotriene CO LO Airway Epithelium Mucu hyperecretion Microvacular leakage Airway mooth mucle Fig. 1. The contribution of different intermediate pathway in airway-narrowing induced by variou indirect timuli. NK: neurokinin receptor; AMP: adenoine 59-monophophate; P: phophate group; AA: arachidonic acid; CO: cyclooxygenae; LO: 5-lipoxygenae. For detail on the different pathway ee the Mechanim and receptor involved in indirect challenge ection of thi report and the report by VAN SCHOOR et al [7]. Mechanim and receptor involved in indirect challenge An overview of the different indirect and direct airway timuli i given in table 1. In figure 1 the contribution of the different intermediate pathway involved in indirect bronchocontriction are outlined. Mechanim involved in the airway narrowing to phyical timuli: evidence from tudie on exercie-induced bronchocontriction Exercie caue airway narrowing by the lo of water via evaporation from the airway urface. The mechanim, whereby the lo of water caue the airway to narrow, i thought to relate to the thermal (cooling and rewarming) [15] and omotic (increae in airway omolarity) effect of dehydration [16]. The dehydration reult in cell hrinkage and lead to a complex equence of biochemical event, a part of the homeotatic repone, producing a retorative increae in the cell volume. For cell uch a the epithelial cell, the mat cell and the enory nerve cell thee biochemical event are likely to timulate the releae of mediator [16]. In-vitro tudie of human lung mat cell how that increaing the omolarity of the olution bathing the cell i a potent timulu to releae of hitamine [17]. The major clinical evidence to upport a role for hitamine releae i the finding that ome hitamine H1 receptor antagonit have an inhibitory effect on exercie-induced bronchocontriction (EIB) [18 20]. Becaue the inhibitory effect i incomplete, hitamine cannot be the only mediator involved in EIB. There are other mat cell mediator that are likely to be involved in EIB, mot notably protaglandin D2 (PGD 2 ) and the cyteinyl leukotriene. Recent tudie have demontrated that the PGD 2 metabolite 9-a 11-b protaglandin F2 i ignificantly increaed in the urine 30, 60 and 90 min potexercie [21, 22]. Thi finding i alo upported by the obervation that flurbiprofen, a cyclooxygenae inhibitor, alo ha a partial inhibitory effect on EIB [19]. Leukotriene are involved in the genei of EIB and in utaining the bronchocontriction following exercie. Repeated tudie have reported increae in urinary leukotriene E 4 following EIB [23, 24]. Some invetigator have alo reported a ignificant increae in leukotriene in bronchoalveolar lavage following dry air hyperpnoea [25]. Alo, there are now many tudie demontrating that both 5-lipoxygenae inhibitor [26 28] and leukotriene receptor antagonit [24, 29] inhibit EIB and enhance recovery of lung function to preexercie value. The inhibition i incomplete confirming that more than one mediator i involved. The epithelial cell i a rich ource of mediator. One uch mediator i protaglandin E2 (PGE 2 ) which may act to protect the airway from narrowing [30]. The releae of PGE 2 may in part be dependent on timulation by leukotriene [8]. Thu, PGE 2 may play an important role in the refractorine that follow exercie. In a recently reported tudy [31], human epithelial cell in culture, when timulated with hypertonic olution rapidly produced interleukin (IL)-8. IL-8 promote

4 INDIRECT AIRWAY CHALLENGES 1053 Table 2. Mediator and neurotranmitter involved in indirect bronchial reponivene Mediator Releae Neuronal Stimulation Reference Adenoine z(hi, LT, PG) z(ach, TK?) [27, 39 49] Tachykinin z(hi, LT, PG) z(ach) [50 56] Bradykinin z(hi, PG, NO) z(ach, TK?) [57 63] Propranolol (Hi) z(ach) [64 68] Metabiulphite/SO 2 z(hi, LT, PG) z(ach, TK?) [47, 69 75] Exercie z(hi, LT, PG) z(ach,tk) [19, 21, 22, 24, 27 30, 34, 76, 77 82] Noniotonic aerool z(hi, LT, PG) z(ach, TK?) [17, 83 88] EVH of dry air z(hi, LT) z(ach, TK?) [26, 30, 89 94] PAF z(lt?) [95] Apirin z(pg, LT)? [96] Allergen z(hi, PG, LT, TK?) [97 103] Hi: hitamine; LT: leukotriene C 4,D 4,E 4 ; PG: protaglandin; ACh: acetylcholine; TK: tachykinin; NO: nitric oxide; EVH: eucapnic voluntary hyperpnoea; PAF: platelet activating factor;?: not known for human airway. Thi table wa modified from [7]. neutrophil chemotactic activity, which ha been reported to be increaed during EIB [32]. Airway enory nerve may alo be affected by alteration in omolarity and cell volume. There i abundant evidence from animal tudie that an increae in omolarity timulate enory nerve. In addition, exercie-induced repiratory water lo can caue coughing in human, an effect that i blocked by inpiring humid air [33]. There i ome evidence to upport the role of tachykinin in EIB; the elective tachykinin neurokininreceptor type-1 (NK 1 ) antagonit FK888 hatened the recovery in lung function to baeline after exercie [34]. EIB i ignificantly inhibited or even completely blocked by ingle doe of nedocromil odium, odium cromoglycate [35], fruemide [36] and by repeated doing with inhaled teroid [37]. Thee drug have no direct effect on airway mooth mucle but reduce the functional activity of mat cell, epithelial cell and enory nerve, implying a ignificant role for thee cell in EIB. The other phyical timuli, noniotonic aerool and eucapnic voluntary hyperpnoea of dry air, work through imilar mechanim (table 2). Mechanim involved in the airway narrowing caued by pharmacological timuli: evidence on adenoine-, tachykinin-, and bradykinin-induced bronchocontriction Several cell and mediator are involved in the airway narrowing due to indirect timuli, thee include epithelial cell, inflammatory cell (incorporating mat cell), nerve cell and blood veel. A ummary i given in figure 1 and table 2 and more detail can be found in a recent review on thi ubject by VAN SCHOOR et al. [7]. The effect exerted by an indirect acting pharmacological agent on the airway differ from timulu to timulu, depending on the target and receptor involved and by the preence of degrading enzyme [7]. Adenoine. Adenoine exert it effect on human cell through interaction with pecific adenoine (P1) receptor, of which four ubtype (A 1,A 2A,A 2B, and A 3 ) have been decribed [104]. The A 1,A 2B, and A 3 receptor have been hown to be involved in variou animal and human model of inflammation. In particular, the potential role of A 2B receptor i being increaingly recognied [105]. The future development of pecific and potent adenoine-receptor agonit and antagonit for ue in vivo in athma will clarify the relative importance of thee receptor [106]. Tachykinin. The airway effect of the tachykinin are mediated via tachykinin NK 1 and NK 2 receptor; there i no evidence for the preence of tachykinin NK 3 receptor in human airway. Subtance P ha the greatet affinity for the NK 1 receptor, wherea neurokinin A ha the greatet affinity for the NK 2 receptor, although there i cro-reactivity [107]. In vitro, tachykinin contrict the mooth mucle of human airway, mainly through tachykinin NK 2 receptor [ ]; in mall and medium ized bronchi, tachykinin NK 1 receptor are alo involved [50, 111]. In vivo, inhaled neurokinin A caue bronchocontriction mainly by indirect mechanim [112]. Both tachykinin NK 1 and NK 2 receptor are involved in the bronchocontrictor effect of neurokinin A [113, 114]. Following their releae from enory cell and immune cell, the tachykinin are degraded by at leat two enzyme: thee are neutral endopeptidae (NEP; EC ) [115, 116] and angiotenin converting enzyme (ACE; EC ). NEP i widely ditributed on a variety of airway cell, and in the airway epithelium. NEP appear to be the mot important enzyme for the breakdown of tachykinin in tiue. ACE, on the other hand, i localied predominantly around the vacular endothelium and therefore degrade intravacular peptide [117]. Bradykinin. Bradykinin caue contraction of the airway by timulation of B2 receptor [57, 58, 118]. Bradykinin i metabolied by everal peptidae, the mot important of which are carboxypeptidae N (kininae I), ACE and NEP [119]. Pretreatment with inhaled N G -monomethyl-l-arginine, a nitric oxide (NO) ynthae inhibitor, ignificantly potentiated airflow limitation in repone to inhaled bradykinin in athmatic; thi ugget that bradykinin activate the NO ynthae pathway, leading to the releae of NO, which in turn counteract the bronchocontrictor repone to bradykinin [120]. The involvement of hitamine and protaglandin in bradykinin-induced airflow limitation appear to be limited [59, 60]. The bronchocontrictor effect of bradykinin i, at leat in part, mediated via cholinergic vagal nerve, ince pretreatment with ipratropium bromide ignificantly reduced airflow limitation in athmatic [60]. Although bradykinin ha been hown to releae tachykinin in guinea-pig airway [ ], concluive evidence for an involvement of tachykinin in bradykinin-induced bronchocontriction in man i lacking [51, 52, 61]. Diagnotic value of the indirect challenge: a comparion with direct challenge Diagnotic value of the direct challenge, hitamine and methacholine Phyician need objective meaurement uch a a bronchial provocation tet, to diagnoe athma [124]. For hitorical

5 1054 G.F. JOOS ET AL. reaon, bronchial reponivene ha been mot commonly aeed uing the direct timuli hitamine and methacholine [125]. Widely ued method include the 2-min tidal breathing method [126, 127], the counted-breath doimeter method [128] which produce comparable reult with appropriate calibration [129] and the portable counted breath technique [130]. The reult are uually expreed a the provocation concentration (or doe) producing a 20% fall in forced expiratory volume in one econd (PC20; PD20; FEV1). Hitamine and methacholine are approximately equivalent on a mg [131], or mmol [132] bai. Bronchial reponivene to hitamine and methacholine (PC20, PD20) i unimodally log-normally ditributed within the population; thi continuou ditribution plu the 95% confidence interval (CI) of repeatability in the range of doubling concentration [133] lead to a ignificant grey area when trying to define a "normal" repone. Inhalation tet have been arbitrarily defined o that the majority of current athmatic are identified generally by a cut-off point that i at the higher end of the borderline range. Bronchial hyperreponivene i conidered to be preent when the hitamine or methacholine PC20 i v8 16 mg?ml -1 [127] or the PD20 i v mmol [130]. Thee arbitrary definition make the tet highly enitive for the detection of hyperreponivene in a pulmonary function laboratory or hopital clinic population. Thi ha been confirmed by a number of tudie documenting enitivity and cloely related negative predictive value of hitamine and methacholine challenge, approaching 100% for clinically current athma (ymptom within previou few day) a oppoed to epidemiologically current athma (ymptom within the pat year) [ ]. By contrat, the pecificity and poitive predictive value of thee challenge for athma ymptom perform le well in the field. For example, the poitive predictive value of hitamine PC20v8 mg?ml -1 for current ymptom of athma in a random ample from the general population wa hown to be well below 50% [137]. When the cut-off point i reduced, the pecificity and poitive predictive value can approach 100% (for example, PC20 v1 mg?ml -1 [127]) but the enitivity and negative predictive value perform poorly [137]. Thu, methacholine and hitamine at a cut-off point of PC20 of 8 16 mg?ml -1 are highly enitive tet and are bet ued to exclude current active dieae a oppoed to the application of the highly pecific cut-off point of PC20 of 1 mg?ml -1, which permit thee tet to confirm dieae. Patient with nonathmatic fixed airflow limitation (chronic airflow limitation, COPD) alo demontrate bronchial hyperreponivene to hitamine and methacholine [ ]. The characteritic are omewhat different in that there i a trong linear relationhip between bronchial hyperreponivene and the obtructive reduction in FEV1 in ubject with chronic airflow limitation. Subject with COPD alo are le hyperreponive than athmatic. However, bronchoprovocation with direct timuli lack pecificity to be able to detect athma in the preence of reting airflow obtruction. Thu, bronchoprovocation with the directly acting timuli, hitamine and methacholine i extremely enitive for current athma ymptom, but lack pecificity both in differentiating athma from normal and athma from chronic airflow limitation. Diagnotic value of indirect challenge Phyical timuli. Exercie challenge. Many comparion of exercie challenge (EIB) with hitamine and methacholine challenge have produced omewhat variable reult. There i a weak, if tatitically ignificant, correlation between EIB and log hitamine, or methacholine PC20 [142, 143]. Exercie challenge, to a preet threhold, i conitently le enitive but more pecific than the direct challenge in differentiating athma from normal [ ]. There are many athmatic with mild bronchial hyperreponivene to direct timuli who have negative exercie challenge but there are individual who have poitive exercie challenge and negative hitamine or methacholine challenge [150]. The imperfect relationhip between EIB and PC20 and the exitence of a number of EIB-poitive, methacholine-negative individual are indicative of the difference in mechanim involved. The fewer tudie in nonathmatic lung dieae are due in part to the difficulty uch individual have in performing exercie challenge. In children, an exercie challenge i better than methacholine at ditinguihing athma from chronic airway diorder uch a cytic fibroi, bronchioliti obliteran, pulmonary ciliary dykineia and bronchiectai [151, 152]. Additional tudie howing that allergen avoidance reulted in a greater improvement in EIB than in methacholine PC20 [153] and that EIB correlate better with marker of inflammation than methacholine PC20 [154], would upport the poibility that EIB may be more clinically relevant than methacholine PC20. The invetigation decribed in the previou paragraph confirm that a poitive exercie challenge i highly pecific to identify clinical athma, but generally i omewhat inenitive to the preence of clinically relevant mild bronchial hyperreponivene. In thi regard, the enitivity-pecificity profile of exercie challenge, reemble that of a hitamine or methacholine PC20 of 1 or 2 mg?ml -1 [137]. There are two poible explanation. Firt, a the phyical timulu affect many cell that are abnormal in athma, it may more readily identify patient with thi dieae than with other airway inflammatory dieae and therefore ha a high pecificity. Secondly, there i a limit to the extent of timulu that can be achieved, due to the technical and afety contraint of exercie, thi prevent maximal airway provocation, reulting in low enitivity. The indication for exercie teting have been ummaried in tatement from the ERS [1] and the American Thoracic Society (ATS) [155]. Exercie may be ued in the following way. 1) In making a diagnoi of EIB in athmatic patient with a hitory of breathlene during or after exertion. 2) To evaluate the ability of performing demanding or lifeaving work (e.g. military, police, or firefighting work) in peron with a hitory uggeting athma. 3) To determine the effectivene and optimal doing of medication precribed to prevent EIB. 4) To evaluate the effect of anti-inflammatory therapy given acutely (e.g. cromone) or chronically (e.g. teroid and leukotriene antagonit). The recommendation for conducting an exercie tet to identify thoe with exercie-induced bronchocontriction have been decribed in detail in both the ERS [1] and the ATS [155] guideline. The recommendation are imilar in both document. In brief the ubject hould exercie for 6 (children, 12 yr) to 8 (adult) min breathing dry air (v25 uc andv50% relative humidity or v10 mg H 2 O?L -1 ) at an intenity to raie the minute ventilation 14 time above the FEV1 and preferably to 21 time the FEV1 (60% maximum voluntary ventilation) for the lat 4 min of exercie. In the abence of a meaure of ventilation the heart rate hould achieve 90% predicted maximum in the lat 4 min of exercie. Value for FEV1 are meaured before and after exercie. Providing the air i dry and the intenity of exercie appropriate it i only neceary to increae the time of exercie, to increae the everity of the airway repone. A reduction in FEV1 of 10% of the pre-exercie value i widely accepted a outide the repone oberved in healthy individual without athma. Eucapnic voluntary hyperpnoea with dry air. Although there are fewer tudie available, the reult are conitent

6 INDIRECT AIRWAY CHALLENGES 1055 with the finding for exercie challenge. Eucapnic hyperpnoea with dry air i more pecific and le enitive than hitamine or methacholine challenge [147, ]. Dry air challenge i clearly more able to eparate athmatic from ubject with chronic airflow limitation than i hitamine challenge [139, 140]. Eucapnic voluntary hyperpnoea (EVH) of dry air containing 5% carbon dioxide (CO 2 ) for 6 min at a ventilation equivalent to 30 time the FEV1 mimic the effect of exercie a decribed above and ha the ame clinical ignificance [156]. A with exercie a 10% reduction in FEV1 i outide the range for healthy ubject without athma [156]. EVH wa recommended to ae winter athlete competing in the Olympic Game in Salt Lake City, a higher level of ventilation could be more eaily achieved during EVH compared with exercie ergometer. Further with EVH it i poible to imulate the condition of exercie (ventilation, duration, inpired air temperature etc.) in a laboratory etting [156]. In contrat to exercie, doe/repone curve can be contructed. Hypertonic aerool. Bronchial reponivene to hypertonic aline challenge correlate better with erum marker of inflammation than bronchial reponivene to methacholine [159]. It improve more than bronchial reponivene to hitamine after a coure of inhaled corticoteroid. A challenge with hypertonic aline i eay to perform and allow contruction of a doe/repone curve [160]. A recently developed highly portable tet uing mannitol capule and a dry-powder inhaler ha hown promie a an indirect challenge with good correlation with the other indirect phyical challenge, exercie, hypertonic aline and hyperventilation [161, 162]. In one tudy, there wa a reaonable correlation between mannitol PD15 and methacholine PC20 [161]. There appear to be no publihed data on comparative enitivity and pecificity. However, ince ome ubject with poitive mannitol tet had mild bronchial reponivene to methacholine, the mannitol inhalation tet may be more enitive than other indirect challenge for detecting mild bronchial reponivene. In a tudy by BRANNAN et al. [162], 22 of the 23 ubject with exercie athma were identified with mannitol and the only ubject who did not repond had a 10% fall in FEV1 to exercie. The major indication for uing hypertonic aerool are to identify bronchial hyperreponivene conitent with active athma or exercie-induced athma and to evaluate bronchial reponivene that will repond to treatment with antiinflammatory drug. In a tudy by RIEDLER et al. [163], children with a hitory of current wheeze were even time more likely to have a poitive repone to hypertonic aline than aymptomatic children. In an occupational tudy in people reponding poitively to the quetion "have you ever had an attack of athma" the mean percentage fall in FEV1 wa 17.6% compared with 5.8% for thoe who reponded negatively [164]. From the evidence to date, it would appear that bronchial reponivene to a hypertonic aerool i conitent with an athma diagnoi. A tet uing a hypertonic aerool i an alternative to exercie or hyperventilation to identify patient with EIB [76, 162, 165, 166]. Although ome patient can have EIB and be negative to hypertonic aline or mannitol, thi i unuual and ha only been found in peron with very mild EIB [162, 163]. A challenge with a hypertonic aerool can be ued in the aement of a patient with a pat hitory of athma that wihe to cubadive. In a tudy uing 4.5% aline to ae potential diver with a pat hitory of athma (uuallyw5 yr), 17% were found to have an abnormal repone, conitent with a diagnoi of current athma [167]. Another indication for the ue of hypertonic aerool may be in the identification of peron with other airway dieae, e.g. chronic airflow limitation or cytic fibroi, who have an athmatic component to their dieae. Many patient with cytic fibroi are conidered to have athma. A ome of the inhaled medication ued in the treatment of cytic fibroi i hyperomolar, it would alo eem important to identify thoe in whom airway narrowing may occur in repone to treatment of their primary dieae [168]. Both hypertonic aline and mannitol increae mucociliary clearance in ubject with athma, bronchiectai and cytic fibroi [ ]. When given daily, hypertonic aline ha been hown to improve lung function in patient with cytic fibroi [172]. Thu a recommendation for ue of a hypertonic aerool a a therapeutic agent may need to be preceded by an inhalational challenge, with the ame hypertonic aerool [173]. A challenge with a hypertonic aerool may alo be indicated in peron with cough-variant athma. Hypertonic aerool can provoke cough [174, 175], o documenting exceive cough in the abence of airway narrowing may indicate that the cough i not due to athma. Further, the cough normally provoked by inhaling hypertonic aline top very quickly within 1 2 min uggeting a form of refractorine to cough in healthy ubject. Finally, a challenge with a hypertonic aerool may be indicated in pregnancy when a patient chooe not to be challenged with a pharmacological agent. The inhalation of hypertonic aline ha been widely ued to induce putum and to collect inflammatory cell and cytokine in athmatic [ ]. What i unique to hypertonic challenge i that it can be ued to document bronchial reponivene at the ame time a collecting putum [179, 180]. Thi make hypertonic challenge attractive for aeing both acute and chronic treatment with corticoteroid. Ditilled water. ALLEGRA and BIANCO [181] performed the firt inhalation challenge with ultraonically nebulied ditilled water (UNDW) in athmatic patient. The technique wa later modified and tandardied by other invetigator [182, 183]. Inhalation of UNDW evoke only a cough in ome normal ubject and a cough and bronchocontriction in athmatic patient [184]. Bronchial repone to UNDW i normally ditributed. Mot athmatic patient develop bronchocontriction after inhaling v2 ml of UNDW [185]. A poitive repone to UNDW i more likely when PD20 methacholine i v2 mmol [185, 186]. Bronchial repone to UNDW correlate poorly with methacholine reponivene [187]. The degree of bronchial reponivene to UNDW i in good concordance with the repone to exercie and to eucapnic hyperpnoea [165]. A refractory period i evident after UNDW in y50% of patient [185, 188]. Refractorine of bronchial airway to UNDW i decreaed by hitamineinduced bronchocontriction [189], the UNDW-induced refractorine cro react with exercie-induced refractorine [190]. Pharmacological timuli. Adenoine. CUSHLEY et al. [191] reported the firt obervation that inhaled adenoine, but not related nucleotide, caued bronchocontriction in patient with athma. Subequently, PHILLIPS et al. [38] have hown that atopic ubject, when compared to nonatopic control, are relatively more reponive to inhaled adenoine and adenoine 59-monophophate (AMP), than they are to methacholine. The airway repone to thee purine may be an index of mat-cell priming, probably through A 2B receptor timulation, linked to mobiliation of intracellular calcium tore. Indeed, naal challenge with AMP elicit rhinitic ymptom and an immediate rie in hitamine level in the lavage fluid with the greatet increae occurring in atopic compared to nonatopic volunteer [192].

7 1056 G.F. JOOS ET AL. Thi indicate that atopy and other condition, where mat cell are primed for mediator releae, are important determinant of enhanced adenoine-induced hitamine releae and that thi repone may be ued a an index of mat cell priming in vivo. The capacity of adenoine to augment mediator releae from mat cell in vivo indicate that adenoine-induced bronchocontriction in athmatic may depend on the tate of airway mat-cell priming and might be ueful a an in-vivo tet for thi. There are limited data available for comparion of enitivity and pecificity of AMP challenge with the directacting timuli. It require y30 time a much AMP a methacholine to induce bronchocontriction. AMP and exercie challenge are better than methacholine challenge for eparating paediatric athma from paediatric "chronic obtructive lung dieae" i.e. AMP and exercie challenge tended to be negative in the children with cytic fibroi, bronchioliti obliteran, ciliary dykineia and bronchiectai [151, 152]. Nonmoking adult with COPD are ignificantly le reponive to inhaled adenoine than nonmoking athmatic, wherea the enitivity to methacholine i imilar in both group [193]. Taken together, thee finding indicate that adenoine challenge may be a ueful tool in the differential diagnoi of athma and COPD in patient of all age in whom the diagnoi i clinically uncertain. Thi i epecially the cae in nonmoker, ince moker with COPD may how AMP reponivene a well [193]. In addition, the pecificity of adenoine bronchoprovocation for athma together with the high repeatability of thi tet could be ueful for epidemiological tudie. Propranolol. On a molar bai, the doe of propranolol required to induce bronchocontriction in patient with athma i y10 15 time larger than methacholine or hitamine [194]. The limited data upport higher pecificity and lower enitivity for propranolol compared to hitamine or methacholine. Propranolol inhalation tet were negative in the majority of ubject with chronic airflow limitation, upporting better pecificity of propranolol challenge for athma [195]. Bronchocontriction induced by propranol i uually le well tolerated by patient compared to that caued by hitamine, methacholine or adenoine. Neverthele no eriou event have ever been reported following propranolol-induced bronchocontriction either in athmatic or in patient with COPD. In addition, propranolol-induced bronchocontriction can be weakly revered by inhaled adrenergic and anticholinergic drug. Metabiulphite, ulphur dioxide. In epidemiological tudie, airway reponivene to the indirect timulu ulphur dioxide (SO 2 ) and the direct timulu methacholine were compared in a ample of 790 adult aged yr. In thi cohort the prevalence of hyperreponivene to SO 2 wa 3.4%. Among the ubject who had hyperreponivene to methacholine, 22.4% had hyperreponivene to SO 2. There wa no ignificant correlation between the degree of hyperreponivene to methacholine and SO 2 [196]. Apirin. While there i no in-vitro tet available for the detection of intolerance to apirin and cro-reacting nonteroidal anti-inflammatory drug (NSAID) in patient with athma, oral provocation with incremental doe of apirin have been ued to diagnoe thi yndrome [197]. However, the challenge procedure i fairly time conuming, potentially dangerou and hould only be performed in a laboratory with coniderable experience of apirin elicited reaction. More recently, the lyine-apirin inhalation challenge introduced by BIANCO et al. [198] ha proven very ueful in identifying apirin-intolerant athmatic ubject [ ]. In a propective comparative tudy, the lyine-apirin challenge wa found to be a enitive a oral provocation, with repect to production of airway obtruction. In a tudy on 22 conecutive patient with a hitory and/or clinical finding uggetive of apirinintolerance (athma, rhinorrhea, naal polypoi) challenge by both route were performed at leat two week apart. A total of 10 ubject developed ignificant bronchocontriction (o20% drop in FEV1) during either challenge, with the ame abolute enitivity for both tet (9/10). Inhalation challenge provoked repone that developed more promptly (within min), were limited to the airway, caued a leer degree of airway obtruction (mean maximal fall in FEV1 29 6% veru 38 16% for oral challenge) and were more eaily revered [200]. In 19 apirin-tolerant control ubject with the ame baeline pulmonary function, inhalation of lyine-apirin caued no ignificant change in FEV1, upporting the pecificity of the tet. Although oral adminitration i neceary for the detection and invetigation of extrapulmonary reaction, inhalation challenge ha the benefit of afety for ue in clinical practice. For reearch purpoe, the afety and good repeatability of inhalation challenge provide a coniderable advantage over oral challenge, particularly ince a ignificant proportion of apirin-intolerant athmatic uffer from moderate-to-evere athma. Report on the repeatability of lyine-apirin challenge [96, 199] have hown that it i repeatable approximately within a ingle doubling concentration or doe difference. With the methodology decribed below, the 95% CI for the difference in reult between two challenge eparated by day wa fold. A poitive provocation repone to inhaled (or oral) apirin reult in a tate of refractorine to further doe of apirin or other NSAID [198]. The refractory period lat between 2 5 day and deenitiation, a well a crodeenitiation, may be retained provided apirin i ingeted within a maximum interval of 48 h. Complete enitivity to apirin and other NSAID, reappear y7 day after the lat expoure to thee drug [202]. Therefore, repeated challenge for diagnoi or reearch purpoe hould be eparated by at leat 1 week. Another pitfall that may produce fale-negative apirin provocation i indicated by obervation that high doe of glucocorticoteroid may mak apirin intolerance [203]. Moreover, it ha been documented that treatment with antileukotriene [96] and almeterol [204] blunt the lyineapirin induced airway repone. The major indication for uing lyine-apirin inhalation challenge i to identify apirin-enitive athmatic patient and to tudy mechanim involved in bronchocontriction elicited by apirin and other NSAID. Direct veru indirect airway challenge to monitor athma The monitoring of ymptom, airflow obtruction and exacerbation i eential to athma management. Regular monitoring by phyician improve health outcome, provided it include monitoring of control of athma, medication and kill at regular interval [205]. Bronchial reponivene can be aeed at regular clinic viit and i related to athma everity and airway inflammation [205]. It ha been demontrated repeatedly that, depite ignificantly improving ymptom and decreaing airway inflammation, inhaled corticoteroid produce, at bet, a modet decreae in bronchial hyperreponivene a meaured by hitamine or methacholine challenge. Thi obervation ha been made in adult [206] a well a children with athma [207]. Depite thee limitation, direct airway challenge may be ueful in the titration of anti-inflammatory therapy [208]. Indeed SONT et al. [208] have reported that a treatment protocol aimed at

8 INDIRECT AIRWAY CHALLENGES 1057 improving bronchial hyperreponivene to methacholine, a well a ymptom and lung function, led to better athma control, fewer exacerbation and reduced chronic airway inflammation. In view of the clinical and phyiological relevance of indirect challenge, it i deirable to deign tudie that compare the improvement in ymptom and marker of airway inflammation induced by anti-athmatic therapy with their effect on direct and indirect airway challenge. The view that bronchial reponivene to adenoine i a more robut marker of dieae activity, in relation to allergic airway inflammation than other nonpecific timuli, uch a hitamine or methacholine, i upported by a number of clinical tudie. In ubject with active allergic rhiniti, bronchial reponivene to AMP, but not methacholine, i trongly correlated to putum eoinophilia [209]. In a large group of patient with athma, PC20 AMP wa more cloely aociated with eoinophilic airway inflammation than PC20 methacholine [11]. A erie of clinical tudie have confirmed the potential utility of AMP in detecting inflammatory change in adult and paediatric athma. Regular treatment with inhaled corticoteroid reult in a ignificantly greater reduction in AMP reponivene compared to that of direct (methacholine and hitamine) and neurally acting timuli (odium metabiulphite and bradykinin) [210, 211]. In keeping with thi, everal tudie have hown that b-agonit caue greater bronchoprotection againt AMP than againt hitamine or methacholine challenge in patient with athma [212, 213]. VAN VELZEN et al. [12] have hown that improvement in clinical athma occurred in a group of 16 allergic athmatic children admitted to a high-altitude clinic. Thi wa believed to be due to the lower allergen level encountered and wa accompanied by a ignificant reduction in bronchial reponivene to AMP but, interetingly, not to methacholine. On the bai of thee obervation, the author believe that adenoine bronchoprovocation may provide an index that could be ued to urvey dieae progreion, monitor therapy and ae prognoi. Omotic timuli, uch a hypertonic (4.5%) aline and mannitol, hold promie for monitoring athma. A challenge with hypertonic aline or mannitol can be ued to ae the everity of athma, the effect of treatment and the compliance with treatment. In a recent tudy in well-controlled athmatic, LEUPPI et al. [214] demontrated that failure of ucceful reduction in teroid could be predicted by reponivene to mannitol. The ue of 4.5% aline, a an indication of everity of athma and need for teroid, i upported by the finding of RODWELL et al. [215]. In their tudy patient with a PD20 to 4.5% aline of o3.0 ml, i.e. thoe with moderate-to-mild athma, were mot likely to become negative to hypertonic aline during treatment with teroid and to plateau in repone to acute adminitration of nedocromil odium. BRANNAN et al. [216] reported imilar finding for mannitol and nedocromil odium. In the tudy of ANDERSON et al. [217] the increae in PD20 to hypertonic aline in repone to 8 week of treatment with budeonide wa predicted by the increae in PD20, following a ingle doe of odium cromoglycate given 10 min before challenge [217]. A negative repone to challenge with 4.5% aline ugget that the peron either doe not have athma, or that their athma i currently under control with treatment. For example, a patient taking budeonide daily for 4 8 week ha a 50% likelihood of becoming negative to challenge with hypertonic aline [160, 215] and to mannitol [218]. Thee finding are in keeping with 50% of the ubject no longer having EIB after treatment with budeonide [37]. By contrat, it i highly likely that the ame people would remain reponive to inhaled hitamine or methacholine [160, 206, 219]. A bronchial challenge with hypertonic aline can be combined with an induction of putum to ae airway inflammation [176, 220]. IN9T VEEN et al. [179] compared provocation with methacholine (PC20), hypertonic aline and putum induction, a outcome parameter in patient with evere athma during teroid withdrawal [179]. During both induced and pontaneouly occurring exacerbation, increaed bronchial reponivene for methacholine wa noted. However, only the induced exacerbation were aociated with increaed bronchial reponivene to hypertonic aline and increaed percentage of putum eoinophil. Repone to indirect challenge can be an intereting outcome parameter in the evaluation of anti-inflammatory treatment by inhaled teroid or leukotriene receptor antagonit. In a comparative tudy on the effect of 4-week treatment period with three different doe of budeonide (100, 200 and 400 mg?day -1 ), PEDERSEN and HANSEN [221] found a doe/repone effect on lung function and EIB, but not on ymptom or peak expiratory flow rate in the evening. Approximately 53% of the maximum effect againt EIB wa achieved by the lowet budeonide doe and y83% by the highet doe. In a tudy on the effect of two doe of fluticaone propionate (100 and 250 mg b.i.d. compared to placebo), the everity of EIB decreaed ignificantly a compared to placebo within 3 week [13]. Thee reduction in EIB did not differ between the two doe and were utained during the tudy period of 6 month. In contrat, reponivene to methacholine improved during the firt 6 week of the treatment with fluticaone, and teadily increaed with time: after 24 week of treatment, the difference in improvement of PD20 methacholine wa 1.6 doe tep for 100 mg fluticaone b.i.d. and 3.3 doe tep for 250 mg b.i.d. The new inhaled teroid cicleonide (50, 200 and 800 mg?day -1 ) reduced reponivene to AMP and eoinophil in induced putum. In contrat to putum eoinophilia, the reduction in reponivene to AMP wa dependent on the doe of inhaled teroid [14]. The tudie that have compared direct and indirect challenge to monitor athma during anti-inflammatory therapy with inhaled corticoteroid and leukotriene-receptor antagonit are ummaried in table 3. Inhaled corticoteroid led to an attenuation of bronchial reponivene to the majority of different timuli, although to different extent, thereby underlining the antiathmatic efficiency of inhaled corticoteroid. All author found a ignificant, although mall reduction in hitamine or methacholine reponivene. Reult were le conitent for bradykinin reponivene and inhalation challenge uing hyperventilation of air which contained SO 2 [211, 222]. It ha even been argued that AMP reponivene, at leat in children, i a more enitive predictor of the effect of anti-inflammatory therapy than bronchial reponivene to methacholine or bradykinin [211]. In a tudy on the effect of a 2-week treatment with oral or inhaled teroid in adult, athmatic patient, PC20 AMP wa found to be more enitive to change in acute airway inflammation compared to PC20 methacholine [228]. Thi would underline the aertion that indirect challenge may be better uited to ae therapeutic efficacy than direct challenge. Following the ame line of reaoning, LEFF et al. [29] demontrated that EIB wa ignificantly attenuated by long-term treatment with a leukotriene receptor antagonit, wherea methacholine reponivene wa not ignificantly reduced. It hould be noted however that the relatively modet benefit of inhaled teroid on direct challenge hould not per e be conidered a a diadvantage. Thi modet benefit may in fact be highly relevant, a part of bronchial reponivene to hitamine or methacholine may not be enitive to teroid, or may require very prolonged therapy. The low repone to teroid may actually be more informative on e.g. remodelling apect, which may be more important for the long-term management and prognoi of the dieae [208].

9 1058 G.F. JOOS ET AL. Table 3. Direct and indirect challenge tet to monitor athma during anti-inflammatory therapy Firt author [ref. no.] Year Compound Duration of treatment week Doe Direct Challenge Indirect Type Reactivity Type Reactivity WIEBICKE et al Salbutamolz 3 0.2/0.5 mg q.i.d Hitamine Q SO 2 Ø [222] BDP Methacholine Q Hypervent. Ø VATHENEN et al Budeonide mg b.i.d. Hitamine Q Exercie Q [223] Cold air hypervent. FULLER et al.[224] 1991 Budeonide mg?day -1 Hitamine Q Bradykinin Q GROOT et al. [225] 1992 BDP mg q.i.d Hitamine Q Dit. water Q O9CONNOR et al Budeonide mg b.i.d. Methacholine Q Metabiulphite Q [210] AMP QQ BOOTSMA et al Fluticaone mg?day -1 Hitamine Q Dit. water Q [226] BDP 1500 mg?day -1 Hitamine Q Dit. water Q DOULL et al. [211] 1997 BDP mg?day -1 Methacholine Ø Bradykinin Ø DU TOIT et al Budeonide mg?day -1 Hitamine Q Hypertonic aline QQ [160] WEERSINCK et al Salmeterol 6 50 mg b.i.d. Methacholine Q Adenoine Q [227] Fluticaone 250 mg b.i.d. Q QQ Salmeterolz 50z250 mg b.i.d. Q QQ Fluticaone LEFF et al. [29] 1998 Montelukat mg?day -1 Methacholine Ø Exercie QQ BDP: beclomethaone dipropionate; Dit. water: ditilled water; Q: modet reduction; QQ: more pronounced reduction; Ø: no change. Ue of indirect airway challenge in epidemiological tudie Quetionnaire are mot frequently ued to diagnoe athma or other repiratory diorder in epidemiological tudie. They may, however, be ubjective and the level of awarene of the condition in the community may influence the pattern of repone. Similar problem may occur with a doctor9 diagnoi of athma. Thee difference in defining repiratory dieae often caue problem with comparion of epidemiological tudie between different population and over time. Thu, an objective marker cloely aociated with dieae like athma i deirable. In the pat, direct-airway challenge uing hitamine and methacholine have been conidered to be more enitive for a diagnoi of athma or athma ymptom, when compared with indirect tet. However, recent laboratory and epidemiological tudie have hown that thi concept might be in quetion. In a laboratory baed tudy of elite ummer athlete HOLZER et al. [150] found that methacholine PD20 had a enitivity of only 36% to identify the athlete with poitive repone to EVH, a urrogate challenge ued to identify exercie-induced bronchocontriction. For thoe 16 ubject poitive to EVH and negative to methacholine the mean SD percentage fall in FEV1 wa % after EVH and the top doe of methacholine the fall in FEV1 wa %. In a field tudy by HABY et al. [229], in which children were tudied with hitamine and exercie, 45% of thoe poitive to a tandardied exercie challenge were negative to inhaled hitamine with reduction in FEV1 to the highet cumulative doe of hitamine beingv10%. A hitamine challenge in 2,363 Autralian choolchildren aged 8 11 yr, yielded a enitivity of 53% and a pecificity of 90% to detect ubject with a diagnoi of athma [230]. Senitivity and pecificity of the hitamine challenge were imilar to enitivity and pecificity of a hypertonic aline challenge and an exercie challenge in another epidemiological tudy in children from the ame country [163]. For many participant in field tudie, particularly children, indirect challenge, involving more natural timuli, are more appealing. Parent will often not allow their child to inhale a pharmacological agent in epidemiological urvey. Conequently, there ha been increaing interet in the ue of indirect airway challenge for epidemiological tudie. Thee tet mainly comprie of the inhalation of noniotonic olution uch a hypertonic aline or ditilled water, hyperventilation of dry air and variou ort of exercie tet. Hypertonic aline challenge i a relatively inexpenive tet that i afe, well tolerated and reproducible. It can be performed readily in the field. It produce few complaint of dryne or irritation of the throat. In a tudy on 500 children, only 1.5 % of participating children felt that they could not continue the challenge becaue of irritation to the throat or cough. Similarly, 1.6 % of the ame ubject were unwilling to complete a free-running exercie tet becaue of fatigue [163]. The hypertonic aline challenge appear to have ome practical advantage compared to exercie challenge in a field tudy. A challenge with hypertonic aline i not dependent on weather condition (temperature, humidity), nor i it influenced by the level of the child9 fitne and it allow for doe increment and meaurement of doe repone curve, making the challenge afer. The EVH challenge i well tandardied [156, 157] but need a pecial ga mixture ource which make it le uitable for field tudie. Safety apect of indirect airway challenge The afety of tandardied hitamine and methacholine challenge tet i recognied all over the world. Previou guideline on provocation challenge have treed the precaution that need to be taken a well a the relative and abolute contraindication for challenge teting [1]. Thee precaution apply alo to indirect airway challenge, and include: laboratory material, peronnel training and written afety protocol. With regard to phyical challenge there i general conenu that tandardied exercie tet are afe [155]. In the literature there i one documented cae of a fatal