Precipitins and specific IgG antibody to

Transcription

1 48 Thorx 1992;47:48-52 Precipitins nd specific IgG ntibody to Aspergillus fumigtus in chest unit popultion Deprtment of Immunology nd Osler Chest Unit, Churchill Hospitl, Oxford OX3 7LJ J A Fux D J Shle D J Lne Address for reprints: Dr Fux Accepted for publiction 9 October 1991 J A Fux, D J Shle, D J Lne Abstrct Bckground The enzyme linked immunosorbent ssy (ELISA) for detecting IgG ntibodies to Aspergillus fumigtus is more sensitive thn the mesurement of Aspergillus precipitins. The reltion of the results from both techniques to the clinicl pttern of disese in lrge unselected group of ptients from lrge referrl centre is unknown. Methods The clinicl reltion of precipitins to Aspergillus fumigtus to clinicl disese ws determined retrospectively in 98 ptients ttending primry referrl centre. Precipitin results were compred with the specific IgG ntibody to A fumigtus in 88 of the ser. Precipitins were determined by the gr gel double diffusion test nd specific IgG ntibody to A fumigtus by quntittive ELISA. Results Precipitins were detected in the unconcentrted serum of 51 ptients. Thirty nine of these hd mycetom or llergic bronchopulmonry spergillosis, 34 hving specific IgG ntibody to A fumigtus more thn the control rnge. Forty seven ptients hd precipitins only fter threefold concentrtion of serum or to only one of the four A fumigtus ntigen extrcts. Most of these hd specific IgG in or ner the control rnge. Thirty of these hd A fumigtus skin test negtive sthm or bronchiectsis, in which spergillus ws probbly not pthogenic. There ws close reltion between the level of ntibody detected by the ELISA nd the number of precipitin lines. Conclusions This study reffirmed the supportive role of spergillus precipitins in the dignosis of pulmonry spergillosis. No dditionl benefit in the routine use of the ELISA ws seen. It lso showed tht cre should be tken in interpreting positive precipitin results from concentrted serum nd tht using severl rther thn one A fumigtus ntigen extrct is helpful for identifying llergic spergillosis. Aspergillus fumigtus is ssocited with mycetom nd llergic bronchopulmonry spergillosis, the mjor non-invsive forms of pulmonry spergillosis." Precipitting ntibody to A fumigtus my be detected in serum in both disorders.?5 Allergic bronchopulmonry spergillosis, the most common cuse of pulmonry eosinophili, my cuse irreversible fibrosis.' Dignosis is mde on clinicl nd rdiologicl criteri nd supported by the finding of immedite skin rectivity, circulting specific IgE ntibody to A fumigtus, nd precipitin lines on gr gel double diffusion in unconcentrted or concentrted serum.46 In these disorders precipitin results re useful nd their interprettion strightforwrd. In routine prctice, however, precipitin test my be requested becuse of suspicion of A fumigtus involvement (s in difficult sthm), positive skinprick test response to A fumigtus, suspicious rdiologicl ppernces, or peripherl blood eosinophili. It is in these circumstnces tht precipitin results with vrying degrees of positivity occur, which prove difficult to interpret in the context of the supposed or eventul dignosis. The gr gel double diffusion method is the stndrd method for detecting Aspergillus precipitins, but it is qulittive test of low sensitivity. Enzyme linked immunosorbent ssy (ELISA) methods for detecting specific IgG ntibodies to A fumigtus hve greter sensitivity."5 Mny comprisons hve been mde between ELISA nd gr gel double diffusion tests, but these hve usully been limited to smll numbers of highly selected ptients. None hs compred the clinicl vlue of the two methods in routine prctice. We undertook this study to determine the mening of positive result in the precipitin test, to compre the gr gel double diffusion nd ELISA methods in primry referrl popultion, nd to evlute the ELISA s routine method for detecting IgG ntibody to A fumigtus. Methods PATIENTS Precipitins to A fumigtus were detected in serum smples from 98 ptients who ttended primry referrl centre during seven yers. The first positive serum precipitin smple ws used in this study. On the bsis of the precipitin results the following four groups were defined: group 1-ptients with three or more precipitin lines in unconcentrted serum; group 2-ptients with one or two precipitin lines in unconcentrted serum; group 3-ptients with precipitins detectble

2 Precipitins nd specific IgG ntibody to Aspergillusfumigtus in chest unit popultion only fter threefold concentrtion of serum; group 4-ptients with precipitins to only one or two of the four ntigen extrcts tested even fter threefold concentrtion of serum nd not to the sme extrct. A group of 18 rndomly selected precipitin negtive ptients ttending the chest clinic served s controls for the ELISA dt. Cse notes were reviewed to determine the dignosis nd collte results of clinic investigtions. In prticulr, we recorded blood eosinophili (>0 5 x 109/1); positive skinprick test responses to A fumigtus nd other common llergens; A fumigtus specific IgE (rdiollergosorbent test (RAST), Phdenzym Phrmci), sputum production, positive cultures, or microscopic ppernces chrcteristic of A fumigtus nd lung function tests (either forced expirtory volume in one second (FEVy), forced vitl cpcity (FVC), or pek expirtory flow (PEF). Chest rdiogrphs were reviewed nd subjects llocted to clinicl groups by DJS without knowledge of their precipitin grouping. Mycetom ws dignosed from the chrcteristic rdiologicl ppernces, consisting of the presence of rdio-opque mss in lung cvity with n ir crescent. Allergic bronchopulmonry spergillosis ws dignosed in ccordnce with ccepted criteri: chest rdiogrphic bnormlities, immedite cutneous rectivity to Aspergillus, incresed totl nd specific IgE ntibodies to Aspergillus, peripherl blood eosinophili, sthm, nd proximl bronchiectsis45 (though bronchogrphy hd not been performed on ll ptients). Bronchiectsis, not ssocited with llergic bronchopulmonry spergillosis, ws dignosed from history of chronic (over three yers) purulent sputum production, bronchogrphs showing distl bronchiectsis, nd n bsence of immedite skin rectivity to A fumigtus. Asthm ws defined on the bsis of history of reversible symptoms of wheeze, chest tightness, nd dyspnoe with documented 15% reversibility of irflow obstruction (FEV1 or PEF) in response to inhled slbutmol (200 pg). AGAR GEL DOUBLE DIFFUSION Ech serum smple ws tested ginst four extrcts of A fumigtus, two from Bencrd, one from the Mycology Reference Lbortory, nd one produced from locl strin. Tble I Distribution of dignoses in the four precipitin groups (number of smples vilble for the enzyme linked immunosorbent ssy in ech group in prentheses) Precipitin group Dignosis Totl Mycetom 11 (11) (11) ABPA 12 (12) 16 (15) 7 (7) 1 (0) 36 (34) Asthm skin test negtive - 4 (3) 8 (7) 9 (7) 21 (17) Bronchiectsis - 2 (1) 5 (5) 8 (7) 15 (13) Other dignoses - 6 (6) 5 (3) 4 (4) 15 (13) Totl (88) ABPA-llergic bronchopulmonry spergillosis. The locl extrct ws produced from surfce culture on dilysble medium t 37 C for four to five weeks.' The mycelil mtt ws homogenised in the culture filtrte, frozen, thwed, nd sedimented t 3000 rev/min. The superntnt ws filtered through membrne filter (0-45,um), dilysed ginst running tp wter nd then ginst two chnges of distilled wter t 4 C, freeze dried, nd used t concentrtion of 20 mg/ml. Four extrcts were used to include s mny ntigens of A fumigtus s possible. After stining nd drying, precipitin lines were counted (JAF) nd recorded s the gretest number recting to ny extrct.'6 Serum smples were stored t 4 C with 0.1% sodium zide until ssyed in the ELISA. ELISA FOR IgG ANTIBODIES TO A FUMIGATUS An ntibody cpture ssy ws developed with n ntigen extrct from loclly isolted strin of A fumigtus. The intr-ssy nd interssy coefficient of vrition for the locl strin ws below 10%. Results were expressed in terms of specific binding index for ech specimen with reference to highly positive lbortory stndrd.'3 This index ws derived s follows: ODX - ODc 100 ODhC-OD,, where ODX is the opticl density of unknown serum, ODh, the men opticl density of highly positive serum, nd OD,c the men opticl density of negtive control serum. STATISTICAL METHODS Dt were nlysed by precipitin group nd clinicl group. The ELISA dt were skewed, so the significnce of differences between groups ws ssessed by the Mnn-Whitney U test. Exct criticl vlues nd p vlues below 0-05 were ccepted s significnt nd re quoted s such to simplify presenttion. Results re expressed s medins with corrected 25% nd 75% interqurtile rnges (Qlc nd Q3c). Results The most frequent dignosis ws sthm, which occurred in 57 of the 98 ptients nd ws evenly distributed between the four precipitin groups (tble 1). The proportion of ptients with llergic bronchopulmonry spergillosis, ll of whom hd sthm, ws much less in group 4-1/I versus 12/12, 16/20, nd 8/15 in groups 1, 2, nd 3 respectively. Mycetom ws found in 11 ptients nd bronchiectsis in 15 ptients. Of the remining 15 subjects, seven hd chronic irreversible irwys obstruction without fetures of sthm; four hd pulmonry fibrosis relted to tuberculosis, srcoidosis, nd nkylosing spondylitis; nd the reminder my hve been colonised by spergillus becuse of ltered lung clernce mechnisms due to crcinom (2) nd severe pneumoni leding to chronic necrotising pulmonry spergillosis (2). 49

3 50 to Tble 2 Specific binding indices for ech precipitin group nd the control group Specific binding index Precipitin Interqurtile lines Medin rnge Group 1 >2 81* Group * Group 3 Concentrted serumt 19t Group 4 Vrible 4t Controls Zero *p < 0-05 between these groups. tdetectble only fter threefold concentrtion of serum. tp < 0 05 between these groups. CLINICAL ASSESSMENTS Aprt from sputum culture nd microscopy for A fumigtus, 83% of ptients hd skin tests, 96% tests for blood eosinophils, nd 71% tests for sputum eosinophils. All sthmtic ptients with negtive skin prick test response to A fumigtus hd specific IgE to Afumigtus determined nd one third hd positive result. A positive skin test response or specific IgE ntibody to A fumigtus ws recorded in ll the ptients with llergic bronchopulmonry spergillosis nd in qurter of the ptients with myectom, but in no other ptients. Three of the spergillus skin test negtive ptients with sthm hd specific IgE ntibody to one or more common llergens nd blood eosinophili of similr mgnitude to tht seen in the ptients with llergic bronchopulmonry spergillosis. Sputum culture for A fumigtus ws performed for only 31 subjects in groups 1 nd 2 nd 19 in groups 3 nd 4. Positive cultures occurred twice s often in groups 1 nd 2 s in groups 3 nd 4. RELATION BETWEEN PRECIPITIN AND ELISA RESULTS The medin specific binding index ws gretest for group 1 nd lowest in group 4. There were eon I - o Group 1 185U.2.m c B CD -0 60"- Q0 40- U) 20- o Group 2 * Group 3 * Group 4 o 0 8 o 0 9 7E o0 0 i0 0 u. n -L- Mycetom ABPA Asthm SPT negtive Qs_- Bronchiectsis 13 Other Comprison of specific IgG ntibody to Aspergillusfumigtus (specific binding index) nd precipitins (groups 1-4) for the mjor dignostic groups. ABPA-llergic bronchopulmonry spergillosis; SPT-skinprick test negtive. 13 Fux, Shle, Lne Tble 3 Specific binding indicesfor the mjor dignostic groups nd the precipitin negtive control serum smples Specific binding index Interqurtile Dignosis Medin rnge nt Mycetom 100* /11 ABPA 43-5* /36 Asthm spergillus negtive /21 Bronchiectsis /15 Others /15 Controls *p < 0-05 in the comprison with ll following groups. tnumber of serum smples vilble for the enzyme linked immunosorbent ssy. ABPA-llergic bronchopulmonry spergillosis. significnt differences between groups 1 nd 2 nd groups 3 nd 4, but not between groups 2 nd 3 or between group 4 nd precipitin negtive controls. Groups 1, 2, nd 3 hd significntly greter specific binding index vlues thn group 4 nd controls, who hd rnge of 1-18 (tble 2). The specific binding index nd precipitin line results ofgroups 1 nd 2 (positive in unconcentrted serum) were significntly relted (r = 0-08, p < 0-001, df = 45). CLINICAL GROUPS Mycetom All ptients with mycetom hd more thn two precipitin lines in unconcentrted serum. The specific binding index vlues were higher thn those found in the other clinicl groups (p < 0"05; tble 3). Allergic bronchopulmonry spergillosis Most ptients with llergic bronchopulmonry spergillosis (28/36) hd precipitins in unconcentrted serum (groups 1 nd 2); the reminder were in group 3, prt from one ptient in group 4. A wide rnge of ntibody levels ws seen by the ELISA. The specific binding index vlues were significntly lower thn those seen in the ptients with mycetom (though individul results overlpped), but the vlues were significntly greter thn those seen in the other clinicl groups (figure, tble 3). The subjects with llergic bronchopulmonry spergillosis in group 3 hd significntly lower vlues thn those in groups 1 nd 2 (medin 30, Qlc 18"3, Q3c 34-8, n = 7, versus medin 50, Qlc 39-8, Q3c 61-3, n = 27; p = 0-05). The ptients in group 3 were lso seprted significntly from ptients with no precipitins nd from ptients with skin test negtive sthm, though individul results overlpped. Nine ptients with llergic bronchopulmonry spergillosis hd no rdiogrphic evidence of disese t the time of ssessment.79 Their medin specific binding index ws less thn tht of the reminder of the group (those with no rdiologicl evidence: medin 24, Qlc 23"8, Q3c 40"3, n = 9; those with rdiologicl evidence: medin 48, Qlc 37.5, Q3c 62-5, n = 23; p < 0-05). Aspergillus skin test negtive sthm Of the 21 ptients with spergillus skin test

4 Precipitins nd specific IgG ntibody to Aspergillusfumigtus in chest unit popultion negtive sthm, only three hd positive skinprick test responses to other ntigens, though 13 hd recorded bsolute peripherl blood eosinophili. Four ptients were in group 2, eight were in group 3, nd nine were in group 4. The ELISA showed low level of specific ntibody to A fumigtus in the serum of 17 of these ptients. The specific binding index vlues in this group did not differ from those of the controls nd the ptients with bronchiectsis nd were lower thn those of the group with llergic bronchopulmonry spergillosis (p < 0-05; tble 3). Bronchiectsis All the ptients with bronchiectsis hd irwys obstruction (FEVI/FVC below 70%) nd negtive skinprick test response to A fumigtus. Most were in precipitin groups 3 nd 4. The ELISA results showed ntibody levels tht were insignificntly different from those of the ptients with skin test negtive sthm nd the precipitin negtive control group. Other dignoses Ptients in this group hd negtive skinprick test responses nd no IgE ntibody to A fumigtus. Of the 15 ptients, six hd one or two precipitin lines nd five hd precipitins fter concentrtion of the serum, but none ws considered to hve spergillosis. The ELISA results showed moderte ntibody levels, though the medin specific binding index of 25 ws less thn tht in the mycetom group (p < 0 05). Discussion This study reviewed the clinicl significnce of precipitins for positive A fumigtus determined by the gr gel double diffusion test in ptients ttending primry referrl chest clinic nd compred these findings with those obtined by n ELISA method. Precipitin groups 1-4 on the gr gel double diffusion test compred closely with the results of ELISA. More thn 90% of individuls with precipitins in unconcentrted serum hd specific binding index significntly bove control vlues. Previous comprisons between gr gel double diffusion nd ELISA hve been bsed on smll numbers of subjects with well defined diseses by ELISA results. Seprtion between serum from control subjects, serum from non-topic subjects, nd serum with one or two precipitin lines hs not lwys been possible."'2 In comprison bsed on 758 serum smples collected from ptients ttending chest clinic 81% (615) were negtive by both gr-gel double diffusion nd ELISA methods nd, of the 127 (19%) positive by ELISA, only 39 were positive by gr gel double diffusion. Only 46 serum smples were from ptients with spergillosis, however.20 It ws concluded tht the ELISA could detect ntibodies ginst non-precipitting ntigenic components of A fumigtus in ddition to the ntibodies detected by gr gel double diffusion.20 Using n optimised ELISA,` we hve found cler reltion between the results of the two methods. The ELISA lso seprted the different precipitin groups, lthough it did not completely distinguish the mycetom nd llergic bronchopulmonry spergillosis groups from the non-spergillosis groups. In this study 43 of the 51 ptients with precipitins in unconcentrted serum hd mycetom or sthm nd 28 of the 32 with sthm hd llergic bronchopulmonry spergillosis. Of the eight ptients with neither myectom nor sthm in groups 1 nd 2, four died soon fter ssessment nd the development of ntibody to Afumigtus ws probbly terminl event relted to impired lung clernce mechnisms. Of the reminder, one hd cystic fibrosis, one ws repetedly precipitin positive, nd further ptient, with specific binding index of 100, hd extrinsic llergic lveolitis, probbly relted to exposure to spergillus while working in stble. The clinicl relevnce of precipitins detected only fter threefold concentrtion of serum my be difficult to determine. In this study only third of ptients in group 3 hd spergillosis. In ptients with llergic bronchopulmonry spergillosis such results hve lwys been considered to be positive. The proportion of precipitin positive ptients with llergic bronchopulmonry spergillosis incresed from 60% to 84% when precipitins in concentrted serum were considered to represent positive result.4 The group 3 ptients with llergic bronchopulmonry spergillosis hd significntly greter mounts of specific IgG ccording to ELISA thn did the precipitin negtive controls, suggesting tht their precipitin results re cliniclly relevnt. Among the ptients with llergic bronchopulmonry spergillosis, those in group 3 hd specific binding index vlues significntly below those in groups 1 nd 2, though the seprtion of most of the seven ptients in group 3 from the precipitin negtive controls nd the sthmtic ptients with negtive skin test response to A fumigtus suggests tht precipitin results in concentrted serum should be considered to be positive when other criteri of llergic bronchopulmonry spergillosis re met. The ELISA suggests tht cre is needed in interpreting the group 4 precipitin results. If precipitin lines re obtined, especilly if wek nd in response to only one extrct in ptient without evidence ofmyectom or llergic bronchopulmonry spergillosis, further smples my be helpful. Ptients with skin test negtive sthm hd lower medin specific binding index thn the ptients with llergic bronchopulmonry spergillosis nd, lthough two thirds hd blood eosinophili, none hd rdiologicl chnges of bronchopulmonry spergillosis. This supports the suggestion tht specific IgE in ddition to specific IgG is needed to produce the chronic inflmmtory chnges seen in the bronchi with llergic bronchopulmonry spergillosis.67 The ptients with bronchiectsis hd been reported to hve precipitins, but none hd positive skinprick test response nd the ELISA confirmed low concentrtions of specific IgG in this group. Poor trcheobronchil clernce mechnisms my hve mde 51

5 52 Fux, Shle, Lne these ptients more exposed to spergillus ntigen thn re norml subjects, leding to the production of precipitting ntibody. The ELISA hd no mjor dvntges over the routine determintion of precipitins in the dignosis of non-invsive pulmonry spergillosis, unless the use of expnded dignostic criteri for llergic bronchopulmonry spergillosis re dopted.56 A comptible history nd rdiologicl ppernces nd positive response to the A fumigtus skinprick test or specific IgE in ptients with precipitins in unconcentrted or threefold concentrted serum my be tken to indicte the presence of llergic bronchopulmonry spergillosis. Wek precipitin rections were not feture of mycetom nd their presence in ptients not meeting other criteri of llergic bronchopulmonry spergillosis rules out ny importnt degree of pulmonry spergillosis. The ELISA used in quntittive form llows within nd between ptient comprisons,"3 wheres the gr gel double diffusion test is semiquntittive, subjective, nd too insensitive for monitoring the progress of llergic bronchopulmonry spergillosis.49 The use of the ELISA my hve further clinicl dvntge in tht the presumed pre-injury phse of llergic bronchopulmonry spergillosis my be dignosed by using ELISAs for A fumigtus specific IgG nd IgE ntibody. This might be of vlue s llergic bronchopulmonry spergillosis my not be dignosed ccording to current stndrd criteri until pprecible loss of lung function hs occurred,467 though there is no evidence tht tretment t this stge reduces lung destruction. We re grteful to Jnssen Phrmceuticl Ltd, UK, for finncil support nd to Miss H Alexnder for preprtion of the mnuscript. 1 Austwick PKC. Fungi nd ctinomycetes. In: Scdding JG, Cumming G, nd Thurlbeck WM, eds. Scientificfoundtion of respirtory medicine. London: Heinemnn, 1981: Hinson KFW, Moon AJ, Plummer NS. Bronchopulmonry spergillosis. A review nd report of eight new cses. Thorx 1953;7: Longbottom JL, Pepys J. Pulmonry spergillosis; dignostic nd immunologicl significnce of ntigens nd c- substnce in Aspergillus fumigtus. J Pthol Bcteriol 1964;88: Mlo JL, Longbottom JL, Mitchell J, Hwkins R, Pepys J. Studies in chronic llergic bronchopulmonry spergillosis-3. immunologicl findings. Thorx 1977;32: Rosenberg M, Ptterson R, Mintzer R, Cooper BJ, Roberts M, Hrris KE. Clinicl nd immunologicl criteri for the dignosis of llergic bronchopulmonry spergillosis.ann Intern Med 1977;86: Ptterson R, Greenberger PA, Hwing JM, Liot JL, Roberts M. Allergic bronchopulmonry spergillosis: nturl history nd clssifiction of erly disese by serologic nd roengenogrphic studies. Arch Intern Med 1986;146: Greenberger PA, Ptterson R. Allergic bronchopulmonry spergillosis nd the evlution of the ptient with sthm. J Allergy Clin Immunol 1988;81: Chpmn BJ, Cpewell S, Gibson R, Greening AP, Crompton GK. Pulmonry eosinophili with nd without llergic bronchopulmonry spergillosis. Thorx 1989;44: Sepulved R, Longbottom JL, Pepys J. Enzyme linked immunosorbent ssy (ELISA) for IgG nd IgE ntibodies of protein nd polyscchride ntigens to Aspergillusfumigtus. Clin Allergy 1979; Greenberger PA, Ptterson R. Appliction of n enzymelinked immunosorbent ssy (ELISA) in dignosis of llergic bronchopulmonry spergillosis. J Lb Clin Med 1982;99: Mishr SK, Flkenberg S, Msihi KN. Efficcy of enzymelinked immunosorbent ssy in serodignosis of spergillosis. J Clin Microbiol 1983;17: Khn ZV, Richrdson MD, Wrnock DW. Evlution of rpid enzyme-linked immunosorbent ssy for IgG ntibodies to Aspergillus fumigtus in the serologicl dignosis of llergic spergillosis. Int Arch Allergy Appl Immunol 1983;72: Shle DJ, Fux JA. The evlution of quntittive enzyme linked immunosorbent ssy (ELISA) for nti Afumigtus IgG. J Immunol Methods 1985;77: Kuffmn HF, vn der Heide S, Beumont F, Blok H, de Vries K. Clss specific ntibody determintion ginst Aspergillus fumigtus by mens of the enzyme-linked immunosorbent ssy. Int Arch Allergy Appl Intmunol 1986;80: Brummond W, Resnick A, Fink JN, Kurup VP. Aspergillus fumigtus-specific ntibodies in llergic bronchopulmonry spergillosis nd spergillom: evidence of polyclonl ntibody response. J Clin Microbiol 1987;25: Fux JA, Stnley VC, Buckley JR, Prtridge BM. A comprison ofdifferent extrcts ofcndidlbicns in gr gel double diffusion techniques. J Immunol Methods 1975;6: McCrthy DS, Simon G, Hrgreve FE. The rdiologicl ppernces in llergic bronchopulmonry spergillosis. Clin Rdiol 1970;21: Mlo JL, Pepys J, Simon G. Studies in chronic llergic bronchopulmonry spergillosis. II Rdiologicl findings. Thorx 1977;32: Mintzer RA, Rogers LF, Kruglik GD, etl. The spectrum of rdiogrphicfindingsinllergicbronchopulmonryspergillosis. Rdiology 1978;127: Kuffmn HF, Beumont F, Meurs H, vn der Heide S, de Vries K. Comprision of ntibody mesurements ginst Aspergillus fumigtus by mens of double diffusion nd enzyme linked immunosorbent ssy (ELISA). J Allergy Clin Immunol 1983;72: