Positive "alveolar" responses to antigen inhalation provocation tests: their validity and recognition

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1 Thorx, 198, 35, Positive "lveolr" responses to ntigen inhltion provoction tests: their vlidity nd recognition DAVID J HENDRICK, ROBERT MARSHALL, JENNIFER A FAUX, AND JOHN M KRALL From the Deprtment of Chest Diseses, Churchill Hospitl nd Rdeliffe Infirmry, Oxford University, Oxford, nd the Deprtments of Medicine nd Community Medicine, West Virgini University Medicl Center, Morgntown, USA ABSTRACT The vlidity of inhltion tests in the investigtion of extrinsic llergic lveolitis ws ssessed from the results of 144 ntigen nd control tests in 31 subjects. A definitive pttern of positive lte responses ws observed. Rections to nebulised bird serum nd droppings in subjects with bird fncier's lung were identicl to rections fter "nturl" exposures in viries or lofts, nd to rections fter "occuptionl" chllenges in subjects with frmer's lung nd mushroom worker's lung. In generl, positive tests were esily recognised subjectivelv from symptoms nd signs pproprite to n influenz-like illness nd undue respirtory effort on exercise. They were ssocited with significnt chnges in six redily vilble objective monitoring mesurements-exercise minute ventiltion (>+ 15%), body temperture (>37-2C), circulting neutrophils (>+ 25/mm3), exercise respirtory frequency (>+ 25%), circulting lymphocytes (. -5/mm3 with lymphopeni), nd forced vitl cpcity (. -15%). These confirmtory monitoring tests hd specificities of pproximtely 95% nd sensitivities of 85-48%. Mesurement of diffusing cpcity, lung volume subdivisions, or resting minute ventiltion/respirtory frequency proved to be too insensitive to be useful, s did usculttion nd chest rdiogrphy. We conclude tht responses tht do provoke significnt chnges in these less sensitive tests re unnecessrily distressing nd, presumbly, unnecessrily hzrdous. The relevnce of n environmentl ntigen to the development of diffuse lveolr disese consistent with extrinsic llergic lveolitis (EAA) is most usefully ssessed from creful clinicl history. Confirmtory investigtions conventionlly depend on serologicl methods-prticulrly gel diffusion precipitin tests.1-9 While such tests usully demonstrte the presence of precipitting ntibody if the ntigen in question is indeed responsible for the disese, this is not lwys the cse.7 12 Conversely, reltively high proportions of subjects similrly exposed, but pprently unffected, my lso give positive serologicl results The ntibody response my consequently reflect exposure more thn disese As yet no lbortory test hs proved cpble of distinguishing ll ffected sub- Presented to the joint meeting of the British nd Americn Thorcic Societies in London, Jnury Address for reprint requests: Dr DJ Hendrick, Tulne University School of Medicine, Pulmonry Diseses Section, 17 Perdido St, NewOrlens, Louisin 7112, USA. jects from ll who re unffected, nd in n pprecible proportion of cses definitive dignosis cnnot be mde from the clinicl nd lbortory fetures lone. This proportion includes, in prticulr, cses of chronic EAA where experimentl periods of ntigen voidnce my produce negligible improvement, cses where experimentl ntigen voidnce is resisted by the subject (or employer) concerned, nd cses where hitherto unrecognised gent is suspected of provoking the disese. Where dignostic uncertinty does persist, mny specilised centres proceed to inhltion provoction tests. A response is considered to be positive if it reproduces the cute clinicl mnifesttions of EAA. Unfortuntely these cute mnifesttions lck specificity. Influenz-like symptoms of mlise, fever, hedche, widespred muscle ching, nd norexi usully msk ny evidence of specificlly pulmonry response, lthough brethlessness nd cough my occur. 415 Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

2 416 Since the provoking gents used in these tests re commonly derived from mouldy vegettion, vin ser or excret, or contminted wter from cooling systems or humidifiers, the significnce of such systemic disturbnces my be questioned. This is prticulrly true of those ptients with the chronic form of EAA, which is not chrcterised by these systemic fetures. Regrdless of the vlidity of inhltion provoction tests in the investigtion of EAA, there re t present no generlly ccepted criteri tht define positive "lveolr" responses. In some studies the development of fever lone hs been considered cceptble confirmtory evidence,5 2 while in others fever occurred in control subjects.2 Although influenz-like symptoms re the most prominent feture of positive rections, they re not lwys reported. Eyckmns nd collegues2' nd Scdding ech described chllenge test with pigeon serum which ppered to provoke fever nd leucocytosis. No other fetures were recorded nd yet in the former cse pigeon breeder's lung (PBL) ws considered to be confirmed, while in the ltter Scdding considered PBL improbble. Objective mesurements of pulmonry function hve not been reported consistently, probbly becuse significnt chnges re not lwys observed. The chrcteristic findings ssocited with positive lveolr rections re restriction of ventiltion (occsionlly with irwys obstruction) nd decresed diffusing cpcity for crbon monoxide (DLCO), but both ventiltion nd DLCO my remin pprently unchnged.5 29 Grvity dependent crckles my develop, but re not commonly reported, nd very few investigtors hve recorded dt regrding the monitoring vlue of chest rdiogrphy The purposes of this study were to exmine the chrcteristics nd the vlidity of "lveolr" responses to ntigen provoction tests, nd to evlute the potentil roles of number of reltively simple nd redily vilble tests in their recognition. Subjects nd methods Sequentil inhltion provoction tests were completed in 29 subjects in whom EAA ws suspected, nd in two control subjects (two of us) so tht definitive result considered to confirm or exclude EAA ws obtined. In six of these subjects there ws lredy convincing evidence of EAA from the histories, physicl exmintions, chest rdiogrphs, lung function studies, precipitin tests, Dvid J Hendrick, Robert Mrshll, Jennifer A Fux, nd John M Krll nd clinicl courses fter ntigen voidnce. With the remining subjects the evidence ws not conclusive. The mjority of the subjects studied were exposed domesticlly to budgerigrs (known s prkeets in North Americ), pigeons, cnries, or poultry nd were possibly ffected by bird fncier's lung (BFL). In four subjects frmer's lung or mushroom worker's lung ws suspected. Most subjects were investigted s outptients, with intervls of week or so between tests. Some were dmitted to hospitl nd hd tests on consecutive dys, though usully not more thn two ntigen chllenges were crried out during ny one dmission. Informed consent ws obtined from the subjects nd the protocol ws pproved by the hospitl ethicl committee. INHALATION PROVOCATION TESTS Nebulised vin ntigens were inhled through fce msk nd delivered vi Wright nebuliser from two sources: vin serum (dilutions of 1/1-1/5 in crbol-sline) for 5-3 minutes, nd extrcts of vin droppings (3-4 mg/ml in crbol-sline) for 5-3 minutes.32 Before ech test modified skin prick test33 ws performed to identify subjects t risk of developing severe immedite hypersensitivity responses. No such subjects were found. Sequentil tests, involving pproximtely fivefold increments in ntigen dose, were crried out until either n unequivoclly positive response ws obtined or the mximum dose (vin serum 1/5 nebulising -5-1 ml of net serum) hd produced no response. We believe this dose is roughly equivlent to 24 hours nturl exposure in n viry or loft of pproximtely 1 birds.32 Filure to respond t this level ws deemed to exclude BFL. The chllenge exposures were crried out in specilly designed cbinet. In generl, the full exposure of ech test ws undertken in three periods of incresing length seprted by intervls of 5-1 minutes. This further reduced the risk of ny undue immedite rection. Control tests were performed under identicl conditions t some point or points in the series using nebulised crbol-sline lone. The identity of ech test extrct ws not reveled to the subject until the whole series of tests ws completed. The colour of ech extrct, which might hve ided its identifiction, ws conceled by the use of blindfold. Nturl exposure tests with vin ntigens were crried out in budgerigr viry nd in two pigeon lofts of locl breeders. Two frm workers nd one mushroom worker produced smples of the hy, strw, nd mushroom compost respect- Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

3 Alveolr responses to provoction tests ively involved, nd disturbed these in the chllenge cbinet environment. One other mushroom worker simply returned to work under our supervision. The durtion of nturl exposures vried from 1 minutes to five hours. MONITORING TESTS Immeditely before ech chllenge, subjects were questioned bout current symptoms nd underwent routine exmintion of the chest. Chllenges were crried out during the morning, usully between 8 nd 1 hours. Subjects were seen t frequent intervls during the next 36 hours, or until ny positive response subsided, nd ny unusul symptoms were recorded. Monitoring investigtions were crried out immeditely before chllenge nd were repeted t lest once lter the sme dy, usully between 16 nd 193 hours. In the event of positive response, the timing of repeted investigtions ws influenced by the time t which symptoms reched their pek. Orl temperture ws mesured immeditely before ech exercise test (see below) nd t hourly intervls throughout the dy. Venous blood ws tken immeditely before ech exercise test. Totl leucocyte counts were mesured in Coulter counter, evening smples being tested the next morning fter storge in refrigertor. Smers were mde from freshly tken blood for differentil cell counts. Pre-chllenge pulmonry function studies included mesurement of forced expirtory volume in one second (FEV1) nd forced vitl cpcity (FVC) using dry Vitlogrph spirometer nd, during the erlier prt of the study, mesurement of DLco by the single breth method34 nd the subdivisions of lung volume by the helium dilution technique.35 Most ptients lerned to use the Vitlogrph spirometer themselves, nd so were ble to record trcings t hourly intervls. In generl, two or three trcings were mde on ech occsion, the one recording the gretest FVC being chosen for nlysis. Becuse erly "positive" tests induced incresed brethlessness on exertion which ws not ssocited with significnt chnges in resting lung function nor with pprecible brethlessness t rest, simple test of lung function during exercise ws devised. This consisted of mesurement of minute volume nd respirtory frequency during three consecutive five-minute periods: sitting t rest; wlking on level tredmill t comfortble constnt rte (one to three miles per hour ccording to individul bility, nd identicl in ll subsequent tests); sitting during recovery. The minute volume ws mesured by brething from dry gs meter, nd the frequency ws recorded by pneumotchygrph in the brething circuit. The significnce of objective chnges ssocited with positive ntigen tests ws ssessed by comprison () of positive ntigen tests with pired control tests in the sme subjects, nd (b) of positive ntigen tests with similr ntigen tests in the subjects shown to be non-rectors. The pired comprisons () were tested sttisticlly with pired t test in the cse of body temperture nd FVC, nd Wilcoxon signed rnks tests in the cse of neutrophil nd lymphocyte counts, exercise minute volume, nd exercise respirtory frequency. Comprisons between ntigen tests (b) were mde with two-smple t test for body temperture nd FVC, nd with Mnn-Whitney U tests for the other mesurements. 36 Results 417 One hundred nd forty-four inhltion provoction tests involving 31 subjects were completed stisfctorily. The result of ech test ws judged t the time s positive, equivocl, or negtive. Positive tests were defined from two stndpoints. Firstly, the subjects concerned developed symptoms of sufficient degree to convince them tht they were directly provoked by the erlier chllenges despite their not knowing whether the nebulised chllenges involved ntigen or control exposures. Secondly, we the observers were lso convinced of the positive nture of the test, tking into ccount not only the chrcter nd timing of the symptoms described, but lso the physicl signs, nd the responses to ny erlier provoction tests. When either prty ws not fully convinced, though suspicious, the result ws defined s equivocl. When neither prty ws suspicious, the result ws deemed negtive. Fifteen ntigen chllenges provoked equivocl results. Fourteen of these occurred in seven subjects who gve unequivoclly positive results fter further chllenges with the sme or higher doses of ntigen. In the remining subject, further ntigen chllenge produced negtive results. A dignosis of EAA (with respect to the ntigen investigted) ws considered to be confirmed in those subjects giving unequivoclly positive responses-"rectors"-nd to be excluded in those who filed to respond in this mnner fter completing the full series of tests-"non-rectors". The two control subjects both proved to be nonrectors, nd the six subjects who lredy showed "convincing evidence" of EAA ll proved to be Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

4 418 rectors. An nlysis of the results is given in tble 1. Positive responses fter ech type of ntigenic chllenge were indistinguishble. The result ctegory of ech test nd the rector sttus of ech subject were consequently determined t the time of the chllenge studies without regrd to the objective monitoring prmeters. The reltive merit of ech potentil monitoring test ws then ssessed by compring the chnges ssocited with positive nd obligtorily negtive tests (ll control tests, nd ll ntigen tests in the non-rectors). Tble 1 Dvid J Hendrick, Robert Mrshll, Jennifer A Fux, nd John M Krll MONITORING TESTS Symptoms Tests ssocited with the development of chrcteristic symptoms tht the subjects regrded s definite re recorded in tble 2. Influenz-like symptoms predominted-mlise, fever, widespred ches nd pins (prticulrly hedche), norexi-nd occurred with ll positive rections. Respirtory symptoms were less striking. Few subjects were troubled by undue brethlessness t rest, though ll noted this on exertion-prticulrly during the exercise test. Severl lso devel- Anlysis of "lveolr" responses to 144 inhltion provoction tests in 31 subjects Subjects Antigen Chllenge exposure nd test result source Control Serum (ml) Droppings (mg) Nturl (hours) < > 5 < >5 < >5 Rectors Avin it B N P 2 B N N N P 3 B N NEE N EP 4t P N NN p p 5 B NNN E N* E EP C P 6 B N N N P 7 B N N PP NN 8 B N N E P P 9t B N N NP N P 1 B NNNN E EE N*P E Y N N lit B N EP 12 B N N P 13 B NN N NN P 14 B N P E 15t P N N N P E Non-vin 16 H N P 17t F N P 18 S N P Non-rectors Avin 19 P N N N N 2 B N N N NN NN 21 B N N N N 22 B N N N N 23 B N N N N 24 B N N N N P P 25 B N N N N 26 B N N N 27 B N N N N 28 B N N N N N 29 B NN N 3 B N N N N N N EN Non-vin 31 M N NN B =budgerigr, C =cnry, F =mushroom frm, H =hy, M =mushroomcompost, P=pigeon, S =strw, Y =poultry, N =negtive, E =equivocl, P=positive. *These two discordnt "negtive" results were obtined in two subjects who gve consistently wek responses to ntigen chllenge irrespective of the chllenge dose. One test ws crried out on the dy immeditely fter n "equivocl" response, nd this my hve exhusted the limited responsiveness ofthe subject concerned. The other subject gve responses tht did not rech their peks until the dy fter chllenge. This ws not pprecited t the time of this nomlous test, nd no subjective dt were recorded for the relevnt following dy. The test did nonetheless produce n objective monitoring score of 2/6 (see text below). tsubjects who lredy showed "convincing evidence" ofeaa before chllenge. Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

5 Alveolr responses to provoction tests Tble 2 Distribution of symptoms nd clinicl signs developing during the 36 hours fter chllenge, ccording to result ctegory Result ctegory Tests Number of tests* ssocited with Symptoms Clinicl signs Flu-like Dysp- Appernce Brething noe Rectors (n = 18) Antigen tests Positive 23 22/ /2 18/18 Equivocl /13 3/1 Negtive 25 1 Control tests 24 2 /23 /23 Non-rectors (n = 13) Antigen tests Control tests 9 *Tests were excluded from tbles 2-4 nd figures I nd 4 whenever the records were incomplete. Corrected totls re given where pproprite. oped dry cough nd difficulty tking deep stisfying breth, but none felt wheezy. In ddition, three rectors developed nuse (one with vomiting) nd two others dirrhoe. In ll but three subjects, symptoms begn fter two to nine hours nd reched mximum intensities fter between six nd 2 hours of the strt of exposure. In one subject with frmer's lung, symptoms begn fter bout one hour, while in two vin rectors the onset of symptoms ws delyed for hours fter the strt of exposure. With both these vin rectors the mximum intensity of the symptoms experienced ws not reched until the next dy. All positive rections were consequently of the "lte" type, nd ll persisted for minimum of severl hours. Clinicl signs Tble 2 gives the distribution of chrcteristic clinicl signs tht the observers regrded s definite, ccording to result ctegory. Fcil ppernce proved to be the most informtive clinicl sign nd the observers soon lerned to recognise the onset of positive responses from the loss of normlly cheerful nd ctive expressions. At this stge the subjects themselves rrely dmitted to ny chnge in well-being. As the response worsened, n unwell ppernce ws generlly redily pprent. Incresed respirtory effort on exercise during the symptomtic phse of positive tests ws usully obvious, but this ws rrely true t rest. Ausculttory signs were uncommon. In five ptients reltively strong rections were ssocited with the development of fine inspirtory crckles in the lung bses. In four ptients, bsl crckles noted before chllenge seemed to become more extensive, nd in three others crckles ppered to develop t the extreme lung bses, but these were not consistently herd. No subject developed wheezes. Body temperture Fever chrcteristiclly ccompnied positive rections nd ws usully symptomtic. The men chnges in body temperture ccording to result ctegory re given in tble 3. Tble 3 Men mximum body tempertures during the 36 hours fter chllenge, ccording to result ctegory Result ctegory Tests Men temperture ( C) per subject* before nd fter chllenge Before After Chnge Rectors (n = 18) Antigen tests Positive Equivocl Negtive Control tests Non-rectors (n=13) Antigen tests Control tests *Becuse individul subjects underwent different numbers oftests, men vlues for ech subject re used in tbles 3 nd 4. Ech subject consequently influenced the results to n equl degree. The results obtined from positive tests in the rectors cnnot be compred in strict sttisticl sense with those from the negtive ntigen tests in the non-rectors becuse different chllenge exposures were used. By considering chllenges using budgerigr serum (BS) lone, the significnce of ny differences ws evluted more stisfctorily. A subgroup of chllenge tests ws selected comprising ll positive tests to BS (1 tests in nine rectors-men nebulised dose, -38 ml net BS), nd ll tests to high dose BS in non-rectors (11 tests in 11 non-rectors-men nebulised dose,.75 ml net BS). These were the tests tht hd determined whether ech budgerigr fncier ws rector or non-rector, respectively. Dt from these selected tests nd the corresponding control tests showed lmost identicl men chnges to those obtined overll (rectors: positive tests +1.5C versus control tests + 3C, p<1; ntigen tests: rectors + 1*5C versus non-rectors +.4C, p<1). This confirmed tht the increse in body temperture ssocited with positive tests ws of sttisticlly significnt degree. Figure 1 compres the distributions of mximum body tempertures fter positive nd obligtorily negtive tests. Mesurements of 37.2C or less were obtined with 95% (73/77) of the obligtorily negtive tests, while mesurements exceeding Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

6 42 Dvid J Hendrick, Robert Mrshll, Jennifer A Fux, nd John M Krll POSITIVE TESTS SENSITIVITY 78% (18/23) - *- ODemo *noun * w Us OBLIGATORILY NEGATIVE TESTS SPECIFICITY 95% (73/77) - O O 37.2 *C N>32'I * DO U OOOOOOOOOOOOU OOOOOOOOOOOOOOOUU MAXIMUM BODY TEMPERATURE ( C) 37-2C were obtined with 78% (18/23) of the positive tests. Body temperture exceeding 37-2C during the 36 hours fter chllenge ws consequently vlid dignostic feture of positive responses, hving high specificity (the dignostic end-point hving been chosen to identify pproximtely 95% of negtives) nd moderte sensitivity (78% of positives identified). Leucocyte counts, exercise tests, nd spirometry Dt on the other objective monitoring prmeters were nlysed in the mnner described for body temperture. Increses in circulting neutro- Fig 1 Distributions of mximl body tempertures during the 36 hours fter chllenge. phil counts, exercise minute volume, nd exercise respirtory frequency, nd decreses in circulting lymphocyte counts nd FVC were found to be sttisticlly significnt nd to hve useful dignostic vlue (sensitivities of pproximtely 5% or more were obtined fter choosing end-points tht produced specificities of bout 95%). The detils re summrised in tble 4. For ech of these mesurements, the subgroups of tests used for sttisticl nlysis showed lmost identicl men chnges to those observed overll. Mrked increses in the numbers of circulting leucocytes were observed in ssocition with Tble 4 Men mximl chnges of objective monitoring tests during the 36 hours fter chllenge, ccording to result ctegory Tests Body Leucocyte counts/mm3 Exercise S minutes % FVC temperture (OC) Neutrophils Lymphocytes* Minute Respirtory % (litres) volume frequency Rectors (n=18) Antigen tests Positive (--61) Equivocl (- 36) Negtive (--16) Control tests (-22) Non-rectors (n = 13) Antigentests (-19) Control tests (- 19) Sttisticl comprisonst Rectors (positive versus p < control tests) Antigen tests (rectors p < versus non-rectors) Dignostic end-point >37-2 >+25 >-5t >+15 >+25 >-15 Specificity 95% 96% 97% 95% 94% 97% Sensitivity 78% 68% 52% 85% 64% 48% *From morning to evening; tusing selected tests with BS nd corresponding control tests; twith lymphopeni. Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

7 Alveolr responses to provoction tests positive tests, nd these chnges were found to be fully ttributble to increses in the neutrophil frction. With 12 of the positive tests bsolute neutrophil (>75 neutrophils/mm3)37 38 developed. The lymphocyte counts were lso ffected by positive tests, though in the converse mnner nd to much less mrked degree. It cn be seen from tble 4 tht the men lymphocyte counts ssocited with control nd negtive tests incresed mildly fter chllenge, while there ws smll decrese ssocited with equivocl tests nd more mrked decrese with positive tests. The bsolute chnges in lymphocyte counts did not prove to be of dignostic vlue when considered lone, becuse there ws considerble overlp between the distributions ssocited with positive nd obligtorily negtive tests. However, decrese in lymphocyte count of 5 cells/mm3 or more did prove to be of dignostic vlue when coupled with the development or worsening of bsolute lymphopeni (<15 lymphocytes/mm3) There were no significnt chnges in the numbers of circulting eosinophils. The results of the exercise tests re expressed in terms of percentge rther thn bsolute chnges becuse there were vritions between the subjects in tredmill speeds nd prechllenge ventiltory ptterns. The exercise period, in comprison with the resting period preceding it nd the recovery period succeeding it, ws ssocited with the gretest increse fter positive tests nd the lest chnge fter obligtorily negtive tests. Mesurement of minute volume nd respirtory frequency during the resting period consequently proved to be much less sensitive in distinguishing positive from obligtorily negtive tests. For both tests n end-point of >±+25% produced specificity of 94%, but sensitivities were only 4% nd 3% respectively. Combining dt from the 1 minutes of exercise + recovery proved to be less useful thn dt from the exercise period lone. With positive responses quite mrked individul vritions in tidl volume nd respirtory rte were noted during the exercise tests, but there ws no overll tendency to rpid shllow brething. The men tidl volume did not chnge significntly nd clcultions with n ssumed ntomicl ded spce of 15 ml showed men increse in lveolr ventiltion of 3% in ssocition with positive tests. There ws men fll of 3% with corresponding control tests. Similr mild men decrements in FVC were noted with ech subgroup of obligtorily negtive tests. These re probbly ttributble to the bis 421 of choosing the minimum of multiple mesurements. The men mximum fll in FVC of 2% fter positive tests ws ccompnied by similr men mximum fll in FEV1 of 19%, indicting the ventiltory disturbnce produced ws one of restriction, not obstruction. The men rtio FEV1/ FVC ws 81% both immeditely before chllenge nd t the time of the mximum fll in FVC. Lung volumes Routine mesurement of the subdivisions of lung volume fter initil positive tests did not demonstrte gret chnges, nd this prctice ws discontinued fter pired positive nd control tests were monitored in four rectors. A modest fll in inspirtory cpcity (men -9%) without chnge in functionl residul cpcity (men -1%) ws the most prominent finding ssocited with positive tests, though mild reduction in expirtory reserve volume (men -7%) ws lso noted. The vitl cpcity nd the totl lung cpcity were reduced ppropritely (mens of -8% nd -5% respectively), nd there ws smll increse in residul volume (men +3%) pproprite to the reduced expirtory reserve volume nd the unchnged functionl residul cpcity. Similr chnges were observed in further rector in whom mesurements fter control chllenge were not obtined. Single breth diffusing cpcity Diffusing cpcity for crbon monoxide ws mesured in ssocition with 12 positive chllenge tests in 1 rectors. Apprecible decreses of 14-45%) occurred with three of the more severe rections only. Figure 2 compres the distributions of chnges in DLco fter positive nd obligtorily negtive tests. Only decrements of 15% or more could be considered dignosticlly significnt, nd with this end-point only two of the positive tests could be identified. Chest rdiogrphs No unequivocl chnges were noted in 12 inspirtory nd five expirtory rdiogrphs of 12 subjects undergoing positive responses. In six films there ws some suspicion of incresed hziness/ nodulrity, for which technicl differences my hve been responsible. TOTAL MONITORING SCORE In ssessing the dignostic vlue of the vrious monitoring tests studied, the bility of ech to identify obligtorily negtive tests correctly (the specificity) ws relted to the converse function of identifying the unequivoclly positive tests Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

8 POSITIVE TESTS -1 1 Dvid J Hendrick, Robert Mrshll, Jennifer A Fux, nd John M Krll I..* OBLIGATORILY NEGATIVE TESTS A % CHANGES IN DLCO Fig 2 Distributions of percentge chnges in DLCO during the 36 hours fter chllenge. correctly (the sensitivity). The endpoints required to give specificities of bout 95% produced useful sensitivity levels (pproximtely 5% or more) for 1 of the tests studied-symptoms nd signs pproprite to n influenz-like illness nd undue respirtory effort on exercise (the four subjective observtions), nd body temperture, circulting neutrophil nd lymphocyte counts, exercise minute volume nd respirtory frequency, nd FVC (the six objective tests). By llowing score of one for ech of these 1 tests giving dignosticlly significnt chnges, mximum totl monitoring score of 8-1 ws possible for the overwhelming mjority of tests (not ll tests were used in ech chllenge). The men scores per chllenge test nd the men scores expressed s percentge of the mximum Tble 5 Totl monitoring scores, ccording to result ctegory Result ctegory Tests Monitoring scores Totl Men Mximum T/M x (T) per possible 1% test (M) Rectors (n =18) Antigen tests Positive Equivocl Negtive Control tests Non-rectors (n= 13) Antigen tests Control tests score possible re shown ccording to result ctegory in tble 5. Obligtorily negtive tests showed, by design, men scores not exceeding 5% of the mximum possible, while positive tests nd equivocl tests (given by the rectors) were ssocited with men scores of 77 nd 31 % respectively. This totl monitoring score proved to be of greter dignostic vlue thn ny of the individul tests considered lone. All the positive chllenge tests were ssocited with scores of 4 or more, while ll the obligtorily negtive tests were ssocited with scores of 3 or less (fig 3). If score of 3 or more is chosen to define positive result, specificity of 99% is obtined. This definition would llow eight of the 15 equivocl tests to be reclssified s "positive." All eight hd occurred in rectors. SEQUELAE In generl the systemic response, s mesured by the influenz-like symptoms, unwell ppernce, temperture increse, nd hemtologicl chnges, subsided spontneously within one to two dys. The pulmonry response, prticulrly incresed brethlessness on exertion nd cough, sometimes persisted longer (in one cse for s much s two to three weeks), nd for this reson the routine use of prednisone (commencing t 3 mg dily nd tiled off over 5-1 dys) ws introduced. With this modifiction, symptoms were generlly stisfctorily controlled within dy or two. The possibility of long-term sequele fter these provoction tests ws considered in 13 of the rectors who were restudied fter intervls rnging from 5 to 33 months (men nine months). Only two considered they hd deteriorted. Pulmonry function tests in the reminder showed tht lung function hd recovered to t lest prechllenge levels, nd often to within the predicted norml rnge. One subject improved initilly when she gve up her birds nd ws treted temporrily with modest doses of corticosteroids (prednisone 2-3 mg dily). Weight gin nd incresed incpcity from severe osteorthritis soon led to reduction in corticosteroid dosge, nd her respirtory symptoms nd lung function therefter worsened stedily. Continuing budgerigr exposure during frequent "bby sitting" ctivities t her son's home ws eventully discovered. One other subject dmitted to mildly incresed brethlessness on exertion when reinvestigted seven months fter her positive chllenge. She hd denied this during the few months immeditely fter it, nd seril lung function studies did not Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

9 Alveolr responses to provoction tests POSITIVE TESTS SENSITIVITY 1% (23/23) _ o EQUIVOCAL TESTS (IN REACTORS) SENSITIVITY 57% o (8/14) o U o o * o U ạ A I.... o oo OBLIGATORILY NEGATIVE TESTS SPECIFICITY - 99% (81/82) o3 < 3 ( TOTAL MONITORING SCORES Fig 3 Distributionv of totl monitoring scores ccording to result ctegory. give confirmtory evidence of deteriorting lung function. It seemed probble tht incresing ngin of effort ws the true cuse of her reduced exercise tolernce. AL Discusson 423 Although inhltion provoction tests re widely used in the investigtion of llergic sthm, their role in the investigtion of EAA is not well estblished. Extrinsic llergic lveolitis is much less common thn sthm, nd mny of the ntigens responsible hve been recognised in recent yers only. Experience is consequently limited. Furthermore, llergic "lveolr" rections re of the "lte" type only, nd so the tests provoking them re prticulrly time-consuming. A more serious problem concerns the vlidity of the positive "lveolr" rection. It could be rgued tht systemic response occurring without evidence of pulmonry dysfunction is cused by the presence of contminting microbil toxins or, fter chllenge with bird serum, by serum sickness; tht systemic hypersensitivity to the test ntigen could hve been induced by sequentil test exposures themselves; nd tht, irrespective of its cuse, systemic hypersensitivity could be unrelted to coexisting lung disese. Tht no significnt responses occurred in the two control subjects or in 11 other non-rectors, is strong evidence ginst toxic mechnisms being directly responsible. Positive tests were reproducible nd were redily distinguished from control tests despite the identity of the nebulised chllenges being unknown to the subjects concerned. Furthermore, positive responses were obtined from ll six of the subjects who hd lredy shown convincing evidence of EAA before undergoing inhltion chllenge. Five of the rectors gve equivocl or positive responses to their first chllenge with the relevnt bird serum, confirming tht in them, t lest, hypersensitivity to bird serum ws not consequence of the test exposures themselves. The three subjects recting to hy, strw, nd mushroom compost respectively lso gve positive responses to their first test exposure. All the postive rections were essentilly identicl in nture, irrespective of the prticulr provoking ntigen or its mens of delivery, nd irrespective of whether the subject concerned mnifested the cute or chronic form of EAA. The responses were shown to involve significnt chnges in lung function, despite the dominnce of systemic disturbnces, nd they closely resembled the cute illnesses with which the pigeon breeders nd frm workers hd presented. We conclude tht the evidence provided by inhltion provoction tests of the type described is both vlid nd definitive in the investigtion of EAA. No other currently vilble test is of equl dignostic vlue. Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

10 424 Subjective observtions found to be useful in monitoring the lveolr response to chllenge were the symptoms nd corresponding clinicl signs of n influenz-like illness nd undue shortness of breth on exercise. All proved to be highly discrimintory, especilly when they were considered together. Their significnce ws limited, however, becuse they were lrgely responsible for estblishing the result ctegory initilly. In this respect the objective prmeters were more vluble, since the objective dt were not nlysed until the study hd been completed nd the result of ech test hd been defined. Six objective mesurements showed sttisticlly significnt chnges in ssocition with positive tests. Overll, they were less sensitive thn the subjective observtions but they confirmed tht significnt systemic nd pulmonry chnges occurred only in the rectors, nd only fter ntigen chllenges. This vlidtes the use of the subjective prmeters in estblishing the result ctegory. Mesurement of minute volume during the exercise period of the exercise test proved to be the most useful objective prmeter, hving sensitivity of 85% (fig 4). This depended to some extent on performing sequentil exercise tests fter chllenge so tht mximl chnges could be Dvid J Hendrick, Robert Mrshll, Jennifer A Fux, nd John M Krll POSITIVE TESTS SENSITIVITY= 85% (7/ 2) * O *n - EE en w l OBLIGATORILY NEGATIVE TESTS SPECIFICITY z 95% o (74/78) O O *U <15% * >, 15% % CHANGES IN EXERCISE MINUTE VOLUME identified. The ltter were found to coincide with the symptomtic peks of positive rections. Mesurement of exercise respirtory frequency proved to be less sensitive thn minute volume but ws, nevertheless, more useful thn ny of the other tests of pulmonry function. Incresed exercise respirtory frequency my hve been prtly dependent on the commonly ccompnying pyrexi, though pyrexi ppered to hve negligible effect on exercise minute volume during het chllenge experiments involving the two control subjects nd well-insulted electriclly heted suit. These divergent effects of fever on respirtion hve been described by Petersen nd Vejby- Christensen.4 In fct, t the times of the relevnt exercise tests, the body temperture of five of the rectors did not exceed 37OOC. Mesurements mde during the initil resting nd subsequent recovery periods of the exercise tests did not in ny combintion improve the dignostic vlue of the exercise period lone. The exercise test s described could consequently be simplified by omitting these two unhelpful periods, nd by performing single follow-up study when ny symptoms pper to be mximl. The exercise test chnges were found to be the result of incresed lveolr ventiltion. It is of s l Fig 4 Distributions of mximl percentge chnges in exercise minute volume during the 36 hours fter chllenge. Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

11 Alveolr responses to provoction tests some interest tht this usully occurred in the bsence of significnt reduction in DLCO. Hyperventiltion nd decresed rteril Pco2 re chrcteristic of more dvnced stges of both cryptogenic fibrosing lveolitis nd EAA, nd it is probble tht hyperventiltion rther thn lrge increses in physiologicl ded spce ws responsible. It my be tht reltively modest diffuse lveolr insults provoke incresed sensitivity of fferent nerve endings nd hence hyperventiltion, when the degree of ny lveolr ventiltion/perfusion imblnce is insufficient to impir crbon monoxide trnsfer. This my lso occur before there is significnt decrese in complince.4142 The usefulness of the exercise test in demonstrting pulmonry involvement in the rections observed my be seen in perspective by considering the other tests monitoring lung function which were ll mesured t rest. Only the repeted mesurement of FVC proved to be worthwhile but its sensitivity ws less thn 5%. Wrren nd collegues used flow volume loops nd single breth nitrogen wshout curves to monitor chnges in lung function during positive "lveolr" responses of six ptients with bird fncier's lung.28 They considered these to be s sensitive s the mesurement of sttic nd dynmic complince, but in generl did not demonstrte ny significnt chnges until the vitl cpcity becme impried. The three remining objective mesurements found to be useful dignosticlly reflected the systemic response. Fever nd neutrophili re both well-recognised fetures of positive "lveolr" responses to provoction tests, nd both proved to hve useful discrimintory vlue. The mild but significnt decrese in the number of circulting lymphocytes is n intriguing finding which hs not been described previously, though Hrgreve nd Pepys observed tht the proportion of lymphocytes decresed with positive tests, while tht of the neutrophils incresed.5 The discrimintory vlue of the lymphocyte chnges ws limited (sensitivity 52%) nd in only one instnce did the lymphocyte count chnge significntly without the neutrophil count doing likewise. Its chief interest concerns its cuse. Could the missing cells hve migrted to the lung s consequence, either of the ntigenic chllenge, or of the resulting immune/inflmmtory response? In its cute phse, EAA is known to be chrcterised by n excess of pulmonry lymphocytes Geller nd collegues hve recently reported low circulting lymphocyte counts in ptients presenting with pulmonry hypersensitivity rections to nitrofurntoin,47 s hve Flherty nd collegues in 425 describing ptients recovering from frmer's lung.48 In both reports it ws the T lymphocyte count which ws reduced, thereby implying involvement of cellulr immunologicl mechnisms. Alterntively, lymphopeni my hve been nonspecific consequence of the stress of the positive response, possibly s result of incresed corticosteroid secretion. Ausculttion, chest rdiogrphy, nd the mesurement of DLCO or the subdivisions of lung volume my be expected to demonstrte significnt chnges only with reltively severe rections. Such rections could be redily identified nywy by the more sensitive monitoring observtions described here. Only six of the 23 positive rections (in five rectors) were ssocited with the development of cler-cut bsl crckles nd with only two of these did the DLco decrese significntly. The lowest totl monitoring score of these six rections ws 5/8, nd the men ws 85% of the totl possible-scores tht comfortbly identify unequivoclly positive responses. Looked t nother wy, inhltion provoction tests tht do produce crckles, significnt flls in DLCO, or rdiogrphic chnges re unnecessrily severe. This implies unnecessry distress to the ptient concerned nd, presumbly, greter hzrd. It is interesting tht the two strongest rections occurred in the two ptients with frmer's lung tested by the nturl method of exposure. They disturbed smples of their own dry mouldy hy nd strw in chllenge cbinet environment for 9 nd 4 minutes respectively. This creted considerbly more dust thn they were ccustomed to occuptionlly nd both rected briskly to the first ctive test. In retrospect, shorter exposures would hve been more pproprite. The vin chllenges involved sequentil incresing doses, the first of which ws known to be sfe.5 The bird fnciers were consequently better protected from unduly strong rections, nd so were less likely to demonstrte chnges with respect to the less sensitive tests. Stronger rections my not lwys be produced by greter chllenge exposures, however, becuse the responsiveness of some subjects is limited. In these circumstnces confirmtion of positive rectivity my require tht wek (though, for the individul concerned, mximl) responses re ppropritely reproducible fter further ntigen nd control chllenges. The device of ssessing ech provoction test by mens of totl monitoring score enbled eight of 15 equivocl tests to be reclssified retrospectively s positive. The subjects concerned might consequently hve been spred the dis- Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

12 426 comfort nd risk of further chllenge exposures. The use of ll six objective mesurements found to be dignosticlly useful ws of course timeconsuming, nd the question rises whether the routine use of ll six would be cost-effective. In sense, none is essentil in evluting the result of most tests becuse the subjective observtions lone were shown to be dequte, being eqully vlid nd rther mnore discrimintory. If, nevertheless, objective evidence is required to confirm the subjective findings, mesurement of exercise minute volume is the most effective of the comprtively simple nd esily vilble monitoring tests considered in this study. It is however the lest convenient nd the most time-consuming. More prcticl is the mesurement of both body temperture nd neutrophil count, one or the other of which is likely to chnge significntly with more thn 9% of positive tests. The costeffectiveness of using ny of the other objective tests in ddition is mrginl. We re grteful to Dr P Green, Dr D Howes, nd Dr H Armitge, Deprtment of Hemtology, Churchill Hospitl, Oxford for crrying out the differentil white blood cell counts, nd to Dr EB Petersen of the Deprtment of Physiology, Oxford University for supplying equipment for the hert chllenge studies. The work ws supported by grnts from the Luder Bequest (Oxford) nd the Oxford Regionl Helth Authority (Teching). This pper contins mteril from thesis submitted to the University of London for the degree of Doctor of Medicine. References Dvid J Hendrick, Robert Mrshll, Jennifer A Fux, nd John M Krll 1 Pepys J, Riddell RW, Citron KM, Clyton YM. Precipitins ginst extrcts of hy nd fungi in the serum of ptients with frmer's lung. Act Allergol 1961; 16: Reed CE, Sosmn A, Brbee RA. Pigeon breeders' lung. JAMA 1965; 193: Cohen HI, Merign TC, Kosek JC, Eldridge F. Sequoiosis. A grnulomtous pneumonitis ssocited with redwood swdust inhltion. Am J Med 1967; 43: Avil R, Villr TG. Suberosis. Respirtory disese in cork workers. Lncet 1968; 1: Hrgreve FE, Pepys J. Allergic respirtory rections in bird fnciers provoked by llergen inhltion provoction tests. Reltion to clinicl fetures nd llergic mechnism. J Allergy Clin Immunl 1972; 5: Blyth W, Grnt IWB, Blckdder ES, Greenberg M. Fungl ntigens s source of sensitiztion nd respirtory disese in Scottish mltworkers. Clin Allergy 1977; 7: Blckburn CRB, Green W. Precipitins ginst extrcts of thtched roofs in the ser of New Guine ntives with chronic lung disese. Lncet 1966; 2: Friend JAR, Gddie J, Plmer KNV, Pickering CAC, Pepys J. Extrinsic llergic lveolitis nd contminted cooling-wter in fctory mchine. Lncet 1977; 1: Fux JA, Hendrick DJ, Annd B. Precipitins to different vin serum ntigens in bird fncier's lung nd coelic disese. Clin Allergy 1978; 8: Skul A. Mushroom-worker's lung. Br Med J 1967; 3: Fux JA, Wide L, Hrgreve FE, Longbottom J, Pepys J. Immunologicl spects of respirtory llergy in budgerigr fnciers. Clin Allergy 1971; 1: Edwrds JH, Bker JT, Dvies BH. Precipitin test negtive frmer's lung--ctivtion of the lterntive pthwy of complement by mouldy hy dusts. Clin Allergy 1974; 4: Pepys J, Jenkins PA. Precipitin (FLH) test in frmer's lung. Thorx 1965; 2: Elmn AJ, Tebo T, Fink JN, Brborik JJ. Rections of poultry frmers ginst chicken ntigens. Arch Environ Helth 1968; 17: Hern CED, Holford-Strevens V. Immunologicl spects of bgssosis. Br J Indust Med 1968; 25: Brborik JJ, Fink JN, Sosmn A, Dhliwl KS. Precipitting ntibody ginst pigeon ntigens in ser of symptomtic pigeon breeders. J Lb Clin Med 1973; 82: Riddle HFV. Prevlence of respirtory symptoms nd sensitiztion by mould ntigens mong group of mlt workers. Br J Indust Med 1974; 31: Fink JN, Schlueter DP, Sosmn AJ et l. Clinicl survey of pigeon breeders. Chest 1972; 62: Slvggio J. Dignostic significnce of serum precipitins in hypersensitivity pneumonitis. Chest 1972; 62: Jckson E, Welch KMA. Mushroom worker's lung. Thorx 197; 25: Eyckmns L, Gyselen A, Luwerijns J, Cosemns J, Wildiers J, Willems J. Pigeon breeder's lung. Dis Chest 1968; 53: Scdding JG. Lung biopsy in the dignosis of diffuse lung disese. Br Med J 197; 2: Scdding JG. Coelic disese nd lung disese. Lncet 1971; 1: Willims JV. Inhltion nd skin tests with exextrcts of hy nd fungi in ptients with frmer's lung. Thorx 1963; 18: Lunn JA, Hughes DTD. Pulmonry hypersensitivity to the grin weevil. Br J Indust Med 1967; 24: Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.

13 Alveolr responses to provoction tests 26 Chnnell S, Blyth W, Lloyd MM et l. Allergic lveolitis in mltworkers. Q J Med 1969; 38: Avil R. Extrinsic llergic lveolitis in workers exposed to fish mel nd poultry. Clin Allergy 1971; 1: Wrren CPW, Chernick RM, Tse KS. Extrinsic llergic lveolitis from bird exposure. Clin Allergy 1977; 7: Boyer RS, Klock LE, Schmidt CD et l. Hypersensitivity lung disese in the turkey rising industry. Am Rev Respir Dis 1974; 19: Hrgreve FE, Pepys J, Longbottom JL, Writh DG. Bird breeder's (fncier's) lung. Lncet 1966; 1: Wrren CPW, Tse KS. Extrinsic llergic lveolitis owing to hypersensitivity to chickenssignificnce of sputum precipitins. Am Rev Respir Dis 1974; 19: Hendrick DJ, Fux JA, Mrshll R. Budgerigrfncier's lung: the commonest vriety of llergic lveolitis in Britin. Br Med J 1978; 2: Hendrick DJ, Dvies RJ, D'Souz MF, Pepys J. An nlysis of skin prick test rections in 656 sthmtic ptients. Thorx 1975; 3: Ogilvie CM, Foster RE, Blkemore WS, Morton JW. A stndrdised breth holding technique for the clinticl mesurement of the diffusing cpcity of the lung for crbon monoxide. J Clin Invest 1957; 36: Comroe JH, Forster RE, Dubois AB, Briscoe WA, Crksen E. The lung. Chicgo: Yer Book Medicl Publictions, Colton T. Sttistics in medicine. Boston: Little, Brown nd Co, Lewis SM. The constituents of norml blood. In: Hrdisty RM, Wetherll DJ (eds). Blood nd its disorders. Oxford: Blckwell Scientific Publictions, 1974: Osgood EE, Brownlee IE, Osgood MW, Ellis DM, Cohen W. Totl, differentil nd bsolute leukocyte counts nd sedimenttion rtes. Arch Intern AMed 1939; 64: Wintrobe MM. Clinicl hemtology. Seventh edition, Phildelphi: Le nd Febiger, 1974: Petersen ES, Vejby-Christensen H. Effect of body temperture on stedy stte ventiltion nd metbolism in exercise. Act Physiol Scnd 1973; 89: Bnszk EF, Thiede WH, Fink JN. Hypersensitivity pneumonitis due to contmintion of n ir conditioner. N Engl J Med 197; 283: Schlueter DP, Fink JN, Sosmn AJ. Pulmonry function in pigeon breeders' disese. Ann Intern Med 1969; 7: Wrren CPW, Tse KS, Chernick RM. Mechnicl properties of the lung in extrinsic llergic lveolitis. Thorx 1978; 33: Moore VL, Fink JN, Brborik JJ, Ruff LL, Schlueter DP. Immunologic events in pigeon breeders' disese. J Allergy Clin Immunol 1974; 53: Reed CE. Allergic mechnisms in extrinsic llergic lveolitis. In: Brent L, Holborow J (eds). Progress in immunology II. Amsterdm: North-Hollnd Publishing, 1974; 4: Royl College of Physicins. Clinicopthologicl conference. Bird fncier's lung. Br Med J 1977; 2: Geller M, Flherty DK, Dickie HA, Reed CE. Lymphopeni in cute nitrofurntoin pleuropulmonry rections. J Allergy Clin Immunol 1977; 59: Flherty DK, Surfus JE, Chmelik F et l. Lymphocyte subpopultions in the peripherl blood of ptients with frmer's lung. Am Rev Respir Dis 1976; 114: Thorx: first published s /thx on 1 June 198. Downloded from on 18 September 218 by guest. Protected by copyright.