protocol-3721.pdf

Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3721|Page 1 of 5Video ArticleDetection of Invasive Pulmonary Aspergillosis in Haematological MalignancyPatients by using Lateral-flow TechnologyChristopher Thornton1,Gemma Johnson2,Samir Agrawal31Biosciences,University of Exeter2BICMS,Queen Mary University of London3St.Bartholomews Hospital and The London NHS TrustCorrespondence to:Christopher Thornton at C.R.Thorntonex.ac.ukURL:http:/ 61,Invasive pulmonary aspergillosis,acute myeloid leukemia,bone marrow transplant,diagnosis,monoclonalantibody,lateral-flow technologyDate Published:3/22/2012Citation:Thornton,C.,Johnson,G.,Agrawal,S.Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by usingLateral-flow Technology.J.Vis.Exp.(61),e3721,doi:10.3791/3721(2012).AbstractInvasive pulmonary aspergillosis(IPA)is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoieticstem cell transplant recipients1.Detection of IPA represents a formidable diagnostic challenge and,in the absence of a gold standard,relies ona combination of clinical data and microbiology and histopathology where feasible.Diagnosis of IPA must conform to the European Organizationfor Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group(EORTC/MSG)consensus defining proven,probable,and possible invasive fungal diseases2.Currently,no nucleic acid-based tests have been externallyvalidated for IPA detection and so polymerase chain reaction(PCR)is not included in current EORTC/MSG diagnostic criteria.Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungalpathogens3,and proven identification requires isolation of the etiologic agent in pure culture.Culture-based approaches rely on the availability ofbiopsy samples,but these are not always accessible in sick patients,and do not always yield viable propagules for culture when obtained.An important feature in the pathogenesis of Aspergillus is angio-invasion,a trait that provides opportunities to track the fungus immunologicallyusing tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage(BAL)fluids.This has led to thedevelopment of the Platelia enzyme immunoassay(GM-EIA)that detects Aspergillus galactomannan and a pan-fungal assay(Fungitell test)that detects the conserved fungal cell wall component(1 3)-D-glucan,but not in the mucorales that lack this component in their cell walls1,4.Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody(MAb)-basedassays that detect surrogate markers of infection1,5.Thornton5 recently described the generation of an Aspergillus-specific MAb(JF5)using hybridoma technology and its use to develop animmuno-chromatographic lateral-flow device(LFD)for the point-of-care(POC)diagnosis of IPA.A major advantage of the LFD is its ability todetect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5.This is animportant consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaledair.The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection,where the LFD displayed improvedsensitivity and specificity compared to the Platelia GM and Fungitell(1 3)-D-glucan assays7.Here,we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids.Its speed and accuracy provides a noveladjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.Video LinkThe video component of this article can be found at http:/ and Preparation of Serum and Bronchoalveolar Lavage Fluids1.Collect serum from untreated blood samples by allowing blood to clot at 4 C,and store serum as aliquots at-20 C prior to use.Bronchoalveolar lavage fluids(BAL)should also be stored as aliquots at-20 C.Note:Storage of serum and BAL as aliquots limits thepotential for degradation of the target antigen by repeated freeze-thawing of samples during repeat testing.2.Thaw samples and mix the serum and BAL samples thoroughly by vortexing and centrifuge for 1 min at 14,000 rpm.Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3721|Page 2 of 53.For routine testing of human serum samples,dilute the serum 1:1(v/v)with tissue culture medium(TCM).TCM consists of RPMI-1640medium,10%(v/v)fetal calf serum,1%(v/v)of 200mM L-glutamine solution,and sodium azide(0.02%w/v)as preservative.The medium isprepared in advance and can be stored at 4 C for several months.Apply 100 l of the diluted serum to the LFD.4.For human BAL fluids,and for BAL fluids from animal models,apply 100 l of neat sample to the LFD,with no pre-treatment.5.For serum from animal models,dilute serum 1:2(v/v)with phosphate buffered saline containing 4%(w/v)EDTA,heat for 3min in a boilingwater bath,centrifuge for 5 min at 14,000 rpm,and apply 100 l of neat supernatant to the LFD device.2.Application of Serum and BAL to the Lateral-flow Device1.Store the lateral-flow devices at room temperature(23 C).At this temperature,devices are stable for 12 months.Remove the devices fromtheir pouches and place on a level surface.2.Using a sterile pipette tip,apply 100 l of pre-treated serum or neat BAL sample to the release port of the device.3.Allow the assay to run for 15 min at room temperature,at which time the results of the tests should be recorded.Note:Within seconds thefluid will be seen to migrate by capillary action along the nitrocellulose membrane in the observation window.3.Recording and Interpreting LFD Results1.The LFD consists of an internal control line(indicated by the letter C on the plastic housing)and a test line(indicated by the letter T).Thecontrol line should always appear irrespective of Aspergillus antigen in the serum or BAL sample.This shows that the assay has run correctly.2.If the Aspergillus antigen is present in the serum or BAL sample,the test line will also appear within 15min of sample application.Becausethe intensity of the test line is proportional to the amount of Aspergillus antigen present in the sample,the test line can appear as a weakpositive(+),a moderate positive(+)or as a strong positive(+).However,any positive test line,regardless of intensity,would indicate thepresence of Aspergillus antigen in the sample.In the absence of Aspergillus antigen,no test line will appear,and the result is recorded asnegative(-).4.Representative ResultsRepresentative examples of negative,weak positive,moderate positive,and strong positive LFD results with BAL and serum samples are shownin Figure 1.Results of antigen-positive LFD tests(weak and strong)or negative LFD tests using BAL fluids from acute myeloid leukemia(AML)patientsdiagnosed according to EORTC/MSG diagnostic criteria are shown in Table 1.Included in this table are the corresponding clinical andmycological(galactomannan and culture)data,and results of an Aspergillus-specific PCR test developed at St.Bartholomews Hospital8,foreach patient.Diagnosis of disease was based on host factors(neutropenia,prolonged use of corticosteroids,treatment with other recognizedT-cell immunosuppressants),clinical criteria and GM positivity for BAL(here defined as a GM index value 0.8).Under the 2002 EORTC/MSGguidelines10,patients 12,13 and 16 were diagnosed with possible IA on the basis of host factors and clinical criteria or GM positivity.Accordingto the revised(2008)EORTC/MSG guidelines2,host factors and GM positivity alone or host factors and clinical criteria alone would not indicatepossible invasive fungal infection unless accompanied by supporting evidence from clinical data and mycology respectively.Patient 6 wasdiagnosed with probable IA under both 2002 and 2008 guidelines because of host factors,major and minor clinical features and GM positivity.Note,that while neither the LFD nor PCR assays are currently included in EORTC/MSG guidelines,there is strong agreement between the twoassays and the commercial galactomannan test indicating the presence of Aspergillus antigen and nucleic acid in the BAL samples of patients 6and 12.Additional results of trials demonstrating the efficacy of the LFD and PCR assays in diagnosing IPA can be found in Johnson et al8.Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3721|Page 3 of 5 Figure 1.Negative(control line only)and positive(control and test line)results of LFD tests using serum and BAL.The intensities of the testline reactions are proportional to the concentrations of the target antigen in the serum and BAL samples.Reactions typically range from weak(+)through moderate(+)to strong(+).Regardless of test line intensity,all three serum positive reactions would indicate invasive pulmonaryaspergillosis disease due to the presence of circulating Aspergillus antigen in the bloodstream.Positive BAL reactions(weak,moderate orstrong)would indicate germination of spores and development of potentially pathogenic hyphae in the lungs.Journal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3721|Page 4 of 5 Patient no.PatientinformationClinicalcriteria1BAL culture2GM EIAindex3EORTC/MSG(2002)4EORTC/MSG(2008)5AspergillusPCR resultLFD result6-Major CTsigns(noduleand halo)andone minorNegative0.9(positive)ProbableProbablePositiveWeak positive(+)12PresumedpreviousfungalinfectionNo major,oneminorCandidaglabrata6.43(strongpositive)PossibleNonePositiveStrong positive(+)13Coagulase-negativeStaphylococcusand E.coli inbloodNo major,twominorNegative0.25(negative)PossibleNoneNegativeNegative(-)16Chest infection 3 minor criteriaincluding newinfiltrate plusplural effusionNegative0.16(negative)PossibleNoneNegativeNegative(-)1 Nodules or halos on a computed tomography scan is suggestive of fungal infection 2 Candida glabrata in BAL fluid would be regarded as a contaminant as it is the second most common yeast isolated as part of normal humanflora9 3 An index of 0.8 in the GM EIA test of BAL is indicative of Aspergillus infection 4 Based on the 2002 EORTC/MSG diagnostic criteria for possible,probable or proven invasive fungal disease10 5 Based on the revised(2008)EORTC/MSG diagnostic criteria for possible,probable or proven invasive fungal disease2Table 1.Results of LFD tests of BAL samples from acute myeloid leukemia patients with probable IPA and from control AML patients(noevidence of infection),and EORTC/MSG diagnosis of infection.DiscussionDefinitive identification of IPA can only truly be achieved by isolation of the etiologic agent from biopsy samples,but recovery of suitable samplesis often not possible in very sick patients and Aspergillus is rarely recoverable from blood.While major advances have been made in the useof computed tomographic scanning of the chest in IPA diagnosis,characteristics that are suggestive of pulmonary IPA such as the haloor air-crescent signs are either transient or can be attributed to breathing artifacts or other fungal infections11,12.Such data are thereforesupplemented with serological techniques that aim to identify signature molecules(GM and-glucan)from fungi that are circulating in thepatients serum or that are present in BAL fluids,sputum or urine samples13.While these tests display satisfactory sensitivity,they lack sufficientspecificity or suffer from interference under certain conditions1,6.The LFD test for IPA detection presented here enables the point-of-care diagnosis of IPA and exploits technology that has been used to date intests for the detection of viruses,bacteria,parasites and toxins14-19 and,most famously,for the home pregnancy tests first introduced by Unipathin 1988.In the Aspergillus LFD described here,the Aspergillus-specific MAb JF5 is immobilized to a capture zone(the test line)on a porousnitrocellulose membrane.Anti-mouse immunoglobulin immobilized to the membrane in a separate zone served as an internal control(controlline).On addition of serum or BAL fluid to the release port,MAb JF5-colloidal gold conjugate in the release pad binds to the target antigen andthe complex then passes along the porous membrane by capillary action.MAb JF5 immobilized in the capture zone binds to the JF5-colloidalgold-antigen complex resulting in a red test line.Any unbound JF5-colloidal gold conjugate binds to the internal control indicating that the assayhas run correctly.This results in a red control line.The test is quick,taking only 15 minutes to perform,is cheap compared to serum and BAL tests based on GM and-glucan detection,anddoes not require expensive equipment or extensive laboratory facilities to run.Furthermore,MAb JF5 does not cross-react with the drugs orcontaminants that have been shown to cause false-positive reaction in the GM and-glucan tests1,4,6.An additional major advantage overcurrent diagnostic tests is the LFDs ability to detect activity that is indicative of invasive growth of Aspergillus species.A critical step in the LFD procedure is the need to read the results 15 min after application of the serum or BAL sample to the device.The testshould not be left for longer than 15 min before recording the results,as this may bias result interpretation.A weak reaction will not be enhancedby extending the incubation period.Heat treatment of serum from animal models of infection has been found to improve assay sensitivity.AJournal of Visualized ECopyright 2012 Creative Commons Attribution-NonCommercial LicenseMarch 2012|61|e3721|Page 5 of 5limitation of the test is that it is qualitative,and relies on the operator to make a subjective assessment of positivity.The intensity of the test linevaries according to the antigen contents of serum and BAL samples(Figure 1).However,any positive reaction(determined by comparison toknown negatives)indicates the presence of Aspergillus antigen and therefore infection.To limit the subjectivity of LFD assays,hand-held devicesare available which allow quantification of LFD test line intensities and enable the establishment of threshold values for antigen detection20.Future developments of the LFD include its commercialization and the development of a multiplex LFD that allows simultaneous detection ofother invasive fungal pathogens using highly specific MAbs3.DisclosuresWe have nothing to disclose.AcknowledgementsFunding to Dr Thornton from Pfizer Limited is gratefully acknowledged.References1.Thornton,C.R.Detection of invasive aspergillosis.Adv.Appl.Microbiol.70,187-216(2010).2.De Pauw,B.,Walsh,T.J.,Donnelly,J.P.,et al.Revised definitions of invasive fungal disease from the European Organization for Researchand Treatment of Cancer/Invasive Fungal Infections Cooperativ