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Quantification of Airborne Aspergillus Allergens: Redefining The Approach

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Quantification of Airborne Aspergillus Allergens: Redefining The Approach
   Journal of Asthma , 47:754–761, 2010Copyright  C   2010 Informa Healthcare USA, Inc.ISSN: 0277-0903 print / 1532-4303 onlineDOI: 10.3109/02770903.2010.492539 ASTHMA AND ENVIRONMENTAL DETERMINANTS Quantification of Airborne  Aspergillus  Allergens: Redefining TheApproach Maansi Vermani, Ph.D., 1 Vannan K. Vijayan, M.D., 2 Mohd A. Kausar, Ph.D., 1 and Mahendra K. Agarwal, Ph.D. 1 , ∗ 1  Department of Respiratory Allergy and Applied Immunology, University of Delhi, Delhi, India 2  Department of Respiratory Allergy and Applied Immunology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India  Background  . Airborne  Aspergillus  species are significant environmental components involved in the pathogenesis and persistence of allergicrespiratory diseases. The detection and quantification of airborne allergens is important to elucidate the clinical implications of environmentalexposure of patients suffering with allergic asthma and/or allergic rhinitis.  Objective . The authors report a simple volumetric approach to measureatmospheric concentration of four common airborne species of   Aspergillus —  A. flavus ,  A. fumigatus ,  A. niger  , and  A. tamarii .  Methods . As particulateaeroallergens may also exist in amorphous form in addition to morphologically identifiable fungal spores/hyphae, a volumetric technique usingmembrane filters was developed for simultaneous quantification of (a) viable  Aspergillus  counts, i.e., colony-forming units (cfu)/m 3 , and (b) actual  Aspergillus  allergen content (ng/m 3 ) in the air. Further, immunochemically quantified airborne  Aspergillus  allergens were compared with theircorresponding colony counts.  Results . The average monthly aerial counts of the four  Aspergillus  species recorded during the sampling year were  A. flavus : 0.25–15.2 cfu/m 3 ;  A. fumigatus : 1.25–15.6 cfu/m 3 ;  A. niger  : 0.75–16.0 cfu/m 3 ; and  A. tamarii : 0.5–11.8 cfu/m 3 of air. Aerial  Aspergillus allergen(s) concentration varied from species to species:  A. flavus : 26.8–680.8 ng;  A. fumigatus : 18.0–380.4 ng;  A. niger  : 28.2–1879.0 ng; and  A.tamarii : 9.2–238.3 ng/m 3 of air. Seasonal distribution of airborne colony counts of the four species didn’t correlate with their respective allergencontent.  Conclusion .  Aspergillus  allergens were present in the air of Delhi area throughout the year with seasonal variations. The authors feel that byusing the immunochemical technique it will be possible to measure actual exposure of patients to various airborne  Aspergillus  allergens. Keywords  Aspergillus ; colony counts; fungi; immunochemical quantification; inhalant allergens Introduction Environmental factors play an important contributory rolein the expression of immunoglobulin E (IgE)-mediated aller-gic diseases such as bronchial asthma and allergic rhinitis,the most important etiologic factor being allergen exposure(1–3). The common inhalant allergens include pollen, fun-gal spores and associated fragments, house dust mite andhouse dust, animal danders, insect emanations and detritus,etc.Thesignificanceoffungalsporesandmycelialfragmentsin the etiology of allergic respiratory disorders has been ex-tensively studied and conclusively established (4–9). Fungalsensitization has been reported to be associated with hospi-tal admissions for asthma, life-threatening exacerbations of asthma, and even death from asthma (9–12).  Aspergillus  species have been reported to be one of themost predominant constituents of aeromycoflora worldwide(4–8, 13–16). Besides conidiospores (2–5  µ m), its hyphalfragments, mostly conidiophores, are also present in the airthroughout the year (17). Once inhaled, they either reachterminal airways or are deposited in large clusters in theupper respiratory tract and cause sensitization, resulting inallergic symptoms. The reported frequency of sensitizationwith  Aspergillus  species in patients with allergic diseaseshas varied from 15.3% to 38% worldwide (13, 18–22). As ∗ Corresponding author: Professor Mahendra K. Agarwal, 90, Anu-pam Apartments, Vasundhra Enclave, New Delhi 110095, India; such, the knowledge of seasonal patterns of occurrence andconcentrations of various  Aspergillus  species in a given geo-graphical area becomes very important for proper diagnosisand management of   Aspergillus  hypersensitive patients.Exposure to fungi including  Aspergillus  species has beentraditionally assessed by gravimetric and volumetric surveyswith the results being reported as spore/colony counts (23,24). However, fungal allergens may also be present in somenonidentifiable submicronic particles such as fungal hyphaeand conidial fragments smaller than intact fungal spores. Onthe other hand, due to the effect of various climatic factorssuch as high temperature, allergenic activity of many viableintactsporesmayhavebeenlost(25,26).Inviewoftheaboveconsiderations, spore counts or viable colony counts maynot be relevant markers of actual airborne allergen contentfor a given fungal species in the air. A more direct way toassess the role of fungal (  Aspergillus ) allergen exposure asa determinant of allergic respiratory diseases is to measureactual allergen concentration in air samples (24–27).Thus, the present study was undertaken for the immuno-chemical measurement of actual total allergen(s) content of four common  Aspergillus  species, viz.  A. flavus ,  A. fumiga-tus ,  A. niger  , and  A. tamarii , in the air and comparison withtheir corresponding viable colony counts. Methods AeromycologicalsurveywasconductedfromMay2007toApril 2008 in Delhi metropolitan area, India (latitude 28 ◦ 4  Nand longitude 77 ◦ 09  E) to study the monthly prevalence of  754  AIRBORNE  ASPERGILLUS   ALLERGENS 755the four species of   Aspergillus . A simple volumetric tech-niqueusingmembranefilterswasdevelopedforsimultaneousquantification of (a) viable  Aspergillus  counts, i.e., colony-forming units (cfu)/m 3 , and (b) actual  Aspergillus  allergencontent (ng/m 3 ) in the air.  Air Sampling Avolumetricsuctionsampler(APM823;EnvirotechLim-ited,India)wasinstalledontheterraceofseven-storeybuild-ing of Vallabhbhai Patel Chest Institute, Delhi, India, at aheight of 20 m above the ground. It was operated for consec-utive 24 hours every week and in total 52 weekly air sampleswere collected during 1 year. The sampler was operated at aflow rate of 40 L/min. A volume totalizer was attached in thesystem to record the volume of the air sampled at any giventime to eliminate the error of fluctuations in the flow rate dueto loading of filters during sampling. The airborne particleswere collected on a polytetrafluoroethylene (PTFE) mem-brane filters (47 mm diameter; 0.45  µ m pore size) mountedon the air sampler in a filter holder assembly. Quantification of Airborne Aspergillus Viable Counts The exposed filters were processed under aseptic condi-tions.Thethinfiltermembraneswithcollectedparticleswerecarefully removed from their support and soaked in 10 ml of sterile extraction buffer (0.1 M NH 4 HCO 3  solution, pH 7.8).The collected particles were removed from the membraneby gentle stirring for 30 minutes resulting in a suspension of airborne particles. This suspension was further used for thesimultaneous quantification of airborne  Aspergillus  countsand allergen content. An aliquot of 0.1 ml of the above sus-pension was plated on culture plates containing Rose Bengalagar medium (in duplicates). The plates were then incubatedat 28 ◦ C ± 2 ◦ C for 3–5 days. The total fungal and  Aspergillus colonies along with the four species—  A. flavus ,  A. fumiga-tus ,  A. niger  , and  A. tamarii —were identified in each plateand counted. The average of duplicate plates was taken forfurther analysis and expressed as colony-forming units perm 3 (cfu/m 3 )ofaircalculatedaspertheformulagivenbelow:cfu / m 3 of air =  No . of colonies in 0 . 1 ml0 . 1 ml  × 10 mlv[1]where v = volume of air sampled (m 3 ).The elution of particles in the remaining suspension (9.8ml) was continued for 72 hours at 4 ◦ C with intermittentshakings.Thesuspensionwasfurtherprocessedforimmuno-chemical quantification ofvariousairborne  Aspergillus  aller-gens described later.  Allergen Extracts and Sera Pools Allergen Extracts.  Crude allergen extracts of the fourspecies of   Aspergillus , viz.  A. flavus ,  A. fumigatus ,  A. niger  ,and  A. tamarii  were purchased from a commercial manufac-turer (All Cure Pharma, Bahadurgarh, Haryana, India). Study Subjects.  A total number of 300 patients (14–60years) suffering with bronchial asthma and/or allergic rhini-tis, diagnosed as per the Global initiative for asthma (GINA)and Allergic Rhinitis and its Impact on Asthma (ARIA)guidelines were included in the present study (2, 28). Thesepatients were selected from the patients attending the OutPatients Department of Viswanathan Chest Hospital, Vallab-hbhai Patel Chest Institute, University of Delhi, Delhi, India.Twenty nonallergic, healthy volunteers (NHVs) with no per-sonal or family history of any allergic disorder were alsoincluded as negative controls. The protocol of the study wasapproved by the Institutional Ethics Committee of Vallabhb-hai Patel Chest Institute, University of Delhi, Delhi, India.Informedconsentwasobtainedfromeachpatientandhealthysubject. Preparation of Sera Pools.  Skin prick tests (SPTs) wereperformed on patients with  Aspergillus  extracts, with 50%glycerinated phosphate-buffered saline (GPBS) and his-tamine (5 mg/ml in GPBS) as negative and positive con-trols, respectively (29). Enzyme allergosorbent test (EAST)was performed to estimate  Aspergillus  allergen–specific IgEin patients’ sera (30). The sera from patients, who showedhighlypositiveSPTaswellasEASTresultstoagivenspeciesof   Aspergillus wereselected( n = 19–23).Fourseparatepoolsof patients’ sera—PPS (  Afl  ), PPS (  Afu ), PPS (  Ani ), and PPS(  Ata ) were prepared using sera of patients hypersensitiveto  A. flavus ,  A. fumigatus ,  A. niger  , and  A. tamarii , respec-tively.ThiswasdonetoensurethepresenceofIgEmoleculesagainstalltheallergenicproteinsofeachextractineachPPS.Another pool of sera from 11 nonallergic healthy subjects(PHS), showing uniformly negative skin test and EAST re-actionstoeach  Aspergillus extract,wasalsopreparedtoserveas a negative control.  Immunochemical Quantification of Airborne  Aspergillus  AllergensProcessing of Air Sample Eluates.  The remaining sus-pensions (after volumetric quantification of   Aspergillus counts) were centrifuged at 4 ◦ C for 10 minutes. The su-pernatants were dialyzed, lyophilized, and reconstituted in1.0 ml of PBS.  Inhibition of   Aspergillus  EAST With Air Sample Eluates. Forquantifyingtheairborne  Aspergillus allergens,airsampleeluates ( n = 52) were used as liquid phase inhibitors (50  µ l)in each  Aspergillus  EAST. In brief, wells of the microtiterplatewerecoatedwith  Aspergillus extract(1 µ g/100 µ l/well)in carbonate buffer (pH 9.5) at 4 ◦ C for 16 hours. The wellswere washed with phosphate-buffered saline–bovine serumalbumin–Tween 20 (PBS-BSA-T) and unoccupied bindingsites were blocked by PBS-BSA. Inhibition of IgE bindingwas attempted by preincubating 50  µ l of PPS (appropriatelydiluted with PBS-BSA-T) with 50  µ l air sample eluate at4 ◦ C for 16 hours. The preincubated mixture was then addedto different wells of a microtiter plate coated with homol-ogous  Aspergillus  extract.  Aspergillus  extract–specific PPS(100  µ l) without any inhibitor and PHS were used as posi-tive and negative controls, respectively. After incubation andwashing of the plate with PBS-BSA-T, 100  µ l of alkalinephosphatase–conjugatedmonoclonalanti-humanIgE(SigmaChemical,St.Louis,MO,USA)inTris-bufferedsaline(TBS;1:1000  v/v ) was added to each well. The plate was then in-cubated at room temperature for 3 hours. Thereafter, thewells were washed with PBS containing 0.05% Tween 20(PBS-T),followedbyadditionof100 µ lofsubstratesolution  756 M. VERMANI ET AL.(1 mg/ml  p -nitrophenyl phosphate in 0.1 M diethanolamine,pH 10.3) in each well. After 30 minutes, the reaction wasstopped by adding 100  µ l of sodium hydroxide (0.75 N).The color developed (optical density; OD) was read by anautomatedmicroplatereaderat405nm.EASTinhibitionwascalculated as follows:Inhibition(%) =  1 − OD of PPS with inhibitorOD of PPS without inhibitor   × 100  Inhibition of   Aspergillus  EAST With Homologous Extract. In addition, to generate a dose-related reference inhibitioncurve, inhibition of each  Aspergillus  EAST was alsoconducted using increasing amounts of correspondinghomologous  Aspergillus  extract as liquid-phase inhibitor.Inhibition of IgE binding was attempted by preincubatingspecific PPS (appropriately diluted with PBS-BSA-T) withincreasing amounts of homologous  Aspergillus  extract (1,10, 100, 1000, 10000 ng of crude extract) at 4 ◦ C for 16hours. The preincubated mixture (100 µ l) was then added todifferent wells of a microtiter plate coated with homologous  Aspergillus  extract. After incubation at 4 ◦ C for 16 hours andwashing of the plate with PBS-BSA-T, 100  µ l of alkalinephosphatase–conjugated monoclonal anti-human IgE inTBS (1:1000  v/v ) was added for 3 hours. Thereafter, thewells were washed with PBS-T followed by addition of 100 µ l of substrate solution. The reaction was stopped after30 minutes by adding 100  µ l of 0.75 N sodium hydroxide.The OD (405 nm) was recorded and inhibition induced byhomologous extract was calculated.A dose-response curve was plotted (amount of allergenextract versus % inhibition). On interpolating the values of % inhibition induced by each air sample eluate in referenceinhibition curve, the amount of allergen extract in 50  µ l of airsampleeluatewasdetermined.Theamountof   Aspergillus allergen (ng/m 3 of air) was calculated as given below:(i) v = Total air sampled in 24 hours (in m 3 )(ii) The eluate of 24-hour air samples was lyophilized andreconstituted in 1000 µ l(iii) Amount of eluate used per well in EAST inhibitionassay = 50 µ lAmount of allergen / m 3 of air =  A llergen in 50 µ l of air samplev  × 20In each assay, specific PPS without any inhibitor and PHSwere usedas positive andnegative controls, respectively. Be-sides, three unrelated heterologous allergen extracts, pollen: Prosopis juliflora , insect: mosquito ( Culex quinquefascia-tus ),andhorsedander,failedtoproducesignificantinhibitionin each  Aspergillus  EAST even when an amount of 10  µ gwasusedasliquidphaseinhibitor,establishingthespecificityof inhibition assays. Specificity of Immunochemical Quantification of   As-pergillus  Allergens.  Specificity of air samples induced in-hibition in different  Aspergillus  EAST was evaluated by per-forming dose related inhibition of each  Aspergillus  EAST bya pool of air samples. For this purpose, equal volumes of 6to 11 air samples showing high allergen content of various  Aspergillus  species were pooled separately to make four sep-arate pools of air samples. Separate inhibition assays of each  Aspergillus  EAST were conducted with increasing amounts(1.56 to 100 µ l) of these specific pools of air sample eluates.As a positive control, dose-related inhibition of each  As- pergillus  EAST was also conducted with homologous  As- pergillus  extract. Slopes of inhibition curves obtained with(a) specific air sample pool and (b) homologous extract ineach  Aspergillus  EAST were compared. As a negative con-trol, extract of a nonexposed filter membrane was also usedin each  Aspergillus  EAST. Statistical Analysis Mean and standard error of the mean ( SEM  ) were deter-mined for data analysis. Spearman correlations (GraphPadPrism version 4.00 for Windows; GraphPad Software, SanDiego, CA, USA; were determined toquantify the relationship (if any) between immunochemi-cally quantified allergen(s) and colony counts of the four  Aspergillus  species in the air of Delhi metropolitan area,India. Results  Airborne  Aspergillus  Viable Counts (cfu/m 3 of air) Average daily airborne fungal count was found to be78.2 cfu/m 3 of air; ranging from 33.0 cfu/m 3 in Decemberto 180.4 cfu/m 3 in March.  Aspergillus  species constituted30.5% of the daily viable fungal counts. Average daily  As- pergillus  counts ranged from 4.2 cfu/m 3 in December to 54.0cfu/m 3 in March.Monthly distribution of average daily colony counts of   A. flavus ,  A. fumigatus ,  A. niger  , and  A. tamarii  in the air duringthesamplingdurationof1yeararepresentedinFigures1Ato4A. The peak periods and range of colony counts of the four  Aspergillus  species during 1 year are presented in Table 1.Of the four  Aspergillus  species, the most prevalent was  A.niger  , accounting for 26.4% of the average daily  Aspergillus counts. The prevalence of   A. tamarii  among the four speciesof   Aspergillus  was relatively low, accounting for 16.6% of the average daily  Aspergillus  counts.  Airborne  Aspergillus  Allergen Content (ng/m 3  of air) Immunochemically quantified airborne  Aspergillus  aller-gen in Delhi metropolitan area showed seasonal variations(Figures 1B to 4B). The peak periods and range of allergencontent of the four  Aspergillus  species during 1 year are pre-sented in Table 1. Of the four  Aspergillus  species,  A. niger  and  A. tamarii  showed the highest and lowest aerial allergenconcentration, respectively.The four pools of air sample eluates produced dose-related inhibition in the corresponding  Aspergillus  EAST(Figures 5 and 6). In each  Aspergillus  EAST, the slopesof these inhibition lines were not statistically differentfrom the inhibition lines produced by respective homol-ogous extract (  p  >  .05). Furthermore, extract of a non-exposed filter membrane used as a liquid phase inhibitor  AIRBORNE  ASPERGILLUS   ALLERGENS 757 F igure  1.—Monthly variations in airborne colony counts (cfu/m 3 ) of   A. flavus and its immunochemically quantified allergen content (ng/m 3 ) in Delhi, India.Bars represent monthly mean ± SEM  . in all the four  Aspergillus , EAST did not show any inhi-bition (Figures 5B and 6B). These results gave evidencefor the specificity of inhibition produced by air sampleeluates. F igure  2.—Monthly variations in airborne colony counts (cfu/m 3 ) of   A. fumi-gatus  and its immunochemically quantified allergen content (ng/m 3 ) in Delhi,India. Bars represent monthly mean ± SEM  . Comparison of Airborne Aspergillus Viable Counts and  Allergen Content  Thehighestviablecolonycountsof   A.flavus ,  A.fumigatus ,  A. niger  , and  A. tamarii  were observed in July, March, May, T able  1.—Airborne  Aspergillus  counts and allergen content in Delhi metropolitan area (May 2007–April 2008). Species Airborne prevalence Highest Lowest Mean Months of peak prevalence Correlation coefficient ∗  A. flavus  Colony counts(cfu/m 3 )July15.2November0.255.1 March–July .0129(p = .9313 # )Allergen content(ng/m 3 )February680.8November26.8181.4 February, October  A. fumigatus  Colony counts(cfu/m 3 )March15.6December1.255.7 March–August  − .1338(p = .3698 # )Allergen content(ng/m 3 )August380.4May18.097.5 August  A. niger   Colony counts(cfu/m 3 )May16.0December0.756.7 March–July .2386(p = .1063 # )Allergen content(ng/m 3 )February1879.0August28.2798.7 February–April, October  A. tamarii  Colony counts(cfu/m 3 )March11.8September0.54.2 March–July .2046(p = .1677 # )Allergen content(ng/m3)August9.2February238.376.7 February, October ∗ Spearman correlation coefficient.#NS = not significant.  758 M. VERMANI ET AL. F igure  3.—Monthly variations in airborne colony counts (cfu/m 3 ) of   A. niger  and its immunochemically quantified allergen content (ng/m 3 ) in Delhi, India.Bars represent monthly mean ± SEM  . andMay,respectively(Table1).Thehighestallergencontentof   A.flavus and  A.niger  wasobservedinFebruaryandthatof   A. fumigatus  and  A. tamarii  in August (Table 1).  A. flavus ,  A. fumigatus ,  A. niger  , and  A. tamarii  viable counts were statis-tically compared with their corresponding airborne allergenconcentration,Spearmancorrelationcoefficients( r  )werenotsignificant (  p > .05) in any comparison (Table 1). Discussion The actual clinical significance of any bioparticle dependson its allergenic potential as well as its presence in the en-vironment of the allergic patients (genetically predisposed).The onset and severity of symptoms of the allergic patientsis related to the appearance and concentration of specific air-borne allergens. Therefore, aeromycological studies givinginformation about fungal allergens of a given geographicalarea and their seasonal patterns are an important supplementfor the diagnosis and treatment of allergic diseases (5, 24,31). This information helps the clinicians in selecting properallergens for diagnostic purposes, providing guidance on al-lergen avoidance and on scheduling medication for allergicpatients (24). F igure 4.—Monthlyvariationsinairbornecolonycounts(cfu/m 3 )of   A.tamarii and its immunochemically quantified allergen content (ng/m 3 ) in Delhi, India.Bars represent monthly mean ± SEM  . In the present study, a volumetric filtration sampler wasused for the quantification of viable  Aspergillus  counts(cfu/m 3 ) in the air. In one of our earlier studies, the volu-metric collection method has been compared with a Burkardspore trap for evaluation of its efficiency and reliability (32).Simultaneous air sampling was conducted with a standardBurkard spore trap at the same site. A significant correlationwas obtained between daily mean concentrations of majorairborne spore types obtained by both the samplers (32).Various aeromycological surveys conducted in differentparts of India from time to time have reported  Aspergillus  tobethemostprevalentconstituent,withconcentrationrangingfrom 5.8% to 33% of total airborne fungi (4, 5, 33–39). Dur-ing our 1-year survey period, colony counts (cfu/m 3 of air)of   Aspergillus  species constituted a predominant componentof total viable airborne fungi (30.5%).Forstudyingallergenicsignificanceof   Aspergillus species,amajorityoftheairsurveysworldwidehaveusuallyenumer-ated them up to genus level only (4, 5, 33, 40–42). However,some  Aspergillus specieshavebeenconsistentlyencounteredin the air surveys conducted in Delhi metropolitan area, suchas  A. flavus ,  A. fumigatus ,  A. niger  , and  A. tamarii  (4, 13).
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