Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

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Diagnostic Microbiology and Infectious Disease

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Clinical utility of Aspergillus galactomannan and PCR in bronchoalveolarlavage fluid for the diagnosis of invasive pulmonary aspergillosis inpatients with haematological malignancies☆

Siow-Chin Heng a, Sharon C.-A. Chen b, C. Orla Morrissey c,d, Karin Thursky e,f, Renee L. Manser g,Harini D. De Silva d, Catriona L. Halliday b, John F. Seymour f,h, Roger L. Nation i,David C.M. Kong a,i, Monica A. Slavin e,f,⁎a Centre for Medicine Use and Safety, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australiab Centre for Infectious Diseases and Microbiology Laboratory Services, ICMPR – Pathology West, Westmead Hospital and the University of Sydney, Darcy Road,Westmead, New South Wales 2145, Australiac Department of Infectious Diseases, Alfred Health and Monash University, Commercial Road, Melbourne, Victoria 3004, Australiad Centre for Biomedical Research, Burnet Institute, 85 Commercial Road, Melbourne, Victoria 3004, Australiae Department of Infectious Diseases, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria 3002, Australiaf Faculty of Medicine, University of Melbourne, Grattan Street, Parkville, Victoria 3010, Australiag Division of Cancer Medicine, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria 3002, Australiah Department of Haematology, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria 3002, Australiai Drug Delivery, Disposition and Dynamic, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia

a b s t r a c ta r t i c l e i n f o

☆ Preliminary data from this work was presented at thon Antimicrobial Agents and Chemotherapy, Denver, CO⁎ Corresponding author. Tel.: +61-3-9656-1707; fax:

E-mail address: monica.slavin@petermac.org (M.A. S

http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.0200732-8893/Crown Copyright © 2014 Published by Else

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Article history:Received 5 October 2013Received in revised form 11 March 2014Accepted 24 March 2014Available online xxxx

Keywords:GalactomannanAspergillus PCRDiagnosisHaematology

Interpretation of Aspergillus galactomannan (GM) and PCR results in bronchoalveolar lavage (BAL) fluid forthe diagnosis of invasive pulmonary aspergillosis (IPA) in patients with haematological malignancies requiresclarification. A total of 116 patients underwent BAL for investigation of new lung infiltrates: 40% wereneutropenic, 68% and 36% were receiving mould-active antifungal agents and β-lactam antibiotics. Thediagnosis of proven IPA (n = 3), probable IPA (n = 15), and possible invasive fungal disease (IFD, n = 50)was made without inclusion of GM results. BAL GM (at cut-off of 0.8) had lower diagnostic sensitivity for IPAthan PCR (61% versus 78%) but higher specificity (93% versus 79%). Both tests had excellent negativepredictive values (85–90%), supporting their utility in excluding IPA. The use of BAL GM and PCR resultsincreased the certainty of Aspergillus aetiology in 7 probable IPA cases where fungal hyphae were detected inrespiratory samples by microscopy, and upgraded 24 patients from possible IFD to probable IPA. Use of BALGM and PCR improves the diagnosis of IPA.

e 53rd Interscience Conference, USA, 2013, abstract M-1273.+61-3-9656-1400.lavin).

vier Inc.

, Clinical utility of Aspergillus galactomannangn Microbiol Infect Dis (2014), http://dx.doi

Crown Copyright © 2014 Published by Elsevier Inc.

1. Introduction

Invasive pulmonary aspergillosis (IPA) is a life-threateninginfection in patients undergoing stem cell transplantation orchemotherapy for haematological malignancies with mortality ratesof 36–75% (Kontoyiannis et al., 2010; Steinbach et al., 2012). Timelyantifungal therapymay improve clinical outcomes, but establishing anearly diagnosis of IPA is difficult (Rano et al., 2001; von Eiff et al.,1995). The current ‘gold standard’ for the diagnosis of proven IPA inthese patients requires histological evidence of tissue infiltration orculture of Aspergillus from biopsy tissue taken from a sterile site(De Pauw et al., 2008). Yet many patients cannot undergo such

invasive procedures, and culture is insensitive (~50%) (Cuenca Estrellaet al., 2011). Non–culture-based serological and molecular diagnosticplatforms have improved sensitivity for the earlier diagnosis of IPA(Mennink-Kersten et al., 2004; White et al., 2010) allowing for moretargeted antifungal therapy.

In haematology patients,Aspergillus galactomannan (GM) enzyme-linked immunosorbent assay (ELISA) andnucleic acid detection by PCRare rapid, sensitive diagnostic tests for IPA (Khot et al., 2008;Maertenset al., 2009). The clinical utility of GM and Aspergillus PCR testing ofserum/blood samples has been confirmed in a randomised controlledtrial (Morrissey et al., 2013). Detection ofAspergillusGMand/orDNA inbronchoalveolar lavage (BAL) fluid may have superior sensitivity overserum/whole blood for IPA as samples are taken from the immediatevicinity of the infection and consequently may have a higher fungalburden. Furthermore, given the time taken for Aspergillus to invadeacross the alveolar capillary barrier [within the initial 24 h after lunginfection as suggested in an in vitromodel (Hope et al., 2007)], testing

and PCR in bronchoalveolar lavage fluid for the.org/10.1016/j.diagmicrobio.2014.03.020

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ofGMand/orDNA inBALfluidmayprovide earlier positive results thantesting on serum. These tests provide a more rapid result than cultureand additionally have better potential to confirm the aetiology of asuspected invasive fungal disease (IFD).

However, considerable variations in the reported test perfor-mance, including sensitivity rates of BAL GM and PCR, have beennoted (Luong et al., 2010; Maertens et al., 2009; Musher et al., 2004;Racil et al., 2011; Reinwald et al., 2012a; Reinwald et al., 2012b), Thesevariations are likely due to differences in patient characteristics andantimicrobial prescribing practices between studies. Additionally, theoptimal interpretive optical density (OD) index cut-off for positivity ofBAL GM has not been fully determined, and different values have beenused between studies (Heng et al., 2013). The lack of standardisationof PCR methods has made it difficult to compare its clinical utility inBAL fluid across studies. Many centres include BAL GM and PCR testingin their clinical management algorithms, but few have reported theirresults, and those that have are single-centre experiences, which maylack generalizability. Therefore, we undertook a study to assess theutility of GM and PCR testing in BAL fluid, for the diagnosis of IPA inhigh-risk haematology patients undergoing diagnostic bronchoscopyin 3 Australian hospitals.

2. Materials and methods

2.1. Patients and samples

Amulticentre retrospective study was conducted between January1, 2007 and December 31, 2012 of all consecutive adult haematologypatients (aged ≥18 years) who underwent diagnostic bronchoscopyfor investigation of pulmonary infiltrates. All included patients hadBAL specimens tested by both Aspergillus GM-ELISA and PCR. MonashUniversity and the Institutional Human Research Ethics Committeesof all study sites approved the study protocol.

All study patients were treated with empirical broad-spectrumβ-lactam at first fever according to National Guidelines (Tam et al.,2011). Bronchoscopy with BAL was performed after detection ofpulmonary infiltrate by radiological evaluation (i.e., by computedtomography [CT] scan, positron emission tomography [PET]/CT (Guyet al., 2012) or, when either was unavailable, chest radiograph). TheBAL procedure typically involved sequential instillation of 3–6aliquots (20 mL each) of sterile saline into the right middle lobe orthe lung sub-segment with themost prominent infiltrate on thoracicimaging (Maschmeyer et al., 2009). BAL sample from the pooledaliquot (discarding the first aliquot for washing) was commonlyused for testing for GM and PCR. Serum GM was not regularlymonitored in all patients, and the result was included in the analysisonly if performed within seven days of BAL.

The following data were extracted for each patient within ±1 month of the bronchoscopy: age, gender, underlying disease, receiptof antifungal agents and β-lactam antibiotics, administration ofchemotherapy prior to bronchoscopy, duration of neutropenia(absolute neutrophil count [ANC] b0.5 × 109/L), and ANC on the dayof bronchoscopy. For adjudication of final diagnosis (see below),results of microbiological evaluations and radiological imaging datewere retrieved.

Patients were classified into 5 categories of likelihood of IPAaccording to the revised European Organisation for the Research andTreatment of Cancer/Mycoses Study Group (EORTC/MSG) consensusdefinitions (De Pauw et al., 2008): proven IPA, probable IPA, possibleIFD, non-Aspergillus invasive mould disease (IMD), or no IFD by 2assessors (M.S. and K.T.) blinded to the GM and PCR results. Anydiscrepancies in case classification were resolved by consensus in ameeting between the 2 assessors and a third member (S.C.H.). Theresults of BAL GM were not included in the classification to avoidincorporation bias. After the initial classification, the BAL GM and PCRresults were revealed to the assessors, and the cases were re-

Please cite this article as: Heng S-C, et al, Clinical utility of Aspergilludiagnosis of invasive pulmonary aspe..., Diagn Microbiol Infect Dis (201

adjudicated incorporating these results, giving equal weight to BALGM and PCR as mycological criteria.

2.2. Platelia® Aspergillus GM assay

The GM-ELISA was performed according to the manufacturer'sinstructions (Platelia® Aspergillus; Bio-Rad, Marnes-la-Coquette,France) (Herbrecht et al., 2002). To define positive GM result forBAL, an OD index cut-off of ≥0.8 was required based on previousfindings (D'Haese et al., 2012; Nguyen et al., 2011). For serumspecimens, an OD index of ≥0.5 was considered positive andconfirmed by re-testing.

2.3. Aspergillus PCR assay

DNA was extracted from 600 μL BAL fluid using a GeneElutemammalian DNA extraction kit (Sigma-Aldrich, St Lois, MO, USA)according to the manufacturer's instructions with a final elutionvolume of 80 μL. A sample of molecular grade water was takenthrough the extraction procedure as a negative extraction control. Anested qualitative real-time PCR assay targeting an Aspergillus genus–specific region of the multi-copy 18S rRNA gene was performed.Oligonucleotide primer pairs (AFU 7S, AFU 7AS; and AFU 5S, AFU 5AS)were used in the first- and second-round PCR amplification reactions,respectively. Reactions were performed in a final volume of 25 μL, aspreviously described (Halliday et al., 2005). For each sample, anegative PCR control of molecular biology grade water and aninhibition control to exclude the presence of inhibitory substanceswere included. Contamination was minimised by performing DNAextraction and PCR amplifications under laminar airflow in 3 separaterooms with unidirectional workflow between all rooms. A specimenwas classified as positive for Aspergillus DNA when there wasexponential increase in fluorescence during the first 30 cycles ofamplification. All positive results were verified by repeat testing.Negative samples had no change in fluorescence. The limit ofdetection of the PCR assay was 0.2 femtogram (fg) of Aspergillus DNA.

All tests were performed by laboratory scientists in 2 laboratories,who were blinded to the clinical condition of the patient. The PCRassays at these 2 laboratories have been cross-validated (Morrisseyet al., 2011).

2.4. Statistical analysis

For the performance analyses and determination of an optimal BALGM OD index (using receiver operating characteristic [ROC] analysis),IPA categories based on the initial classification were used (i.e., BALGM and PCR results were not included as mycological criteria forprobable cases). Proven and probable IPA cases were grouped as true-positive, whereas the true-negative control group consisted of caseswith no IFD. Diagnostic performance in terms of sensitivity,specificity, positive predictive value (PPV) and negative predictivevalue (NPV), positive and negative likelihood ratios, and diagnosticodds ratio, along with their associated 95% confidence intervals (CIs),were calculated. The effects of mould-active antifungal agents,neutropenia, and β-lactam antibiotics on the tests' sensitivity orspecificity were assessed. The Mann-Whitney U-test was used tocompare continuous variables, and Fisher's exact test, for categoricalvariables. The significance level was set at a 2-sided P-value of 0.05.Statistical analyses were conducted using PASW statistical softwareversion 19.0 (IBM SPSS, Inc, NY, USA).

3. Results

A total of 116 BAL samples from 116 haematology patients withpulmonary infiltrates or nodules were submitted for GM and PCRtesting between January 1, 2007, and December 31, 2012. Of the

s galactomannan and PCR in bronchoalveolar lavage fluid for the4), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.020

Table 1Baseline patient characteristics.

Characteristics Proven/probable(n = 18)

No IFD(control, n = 42)

Age: years; median (range) 54 (19–80) 59 (18–72)Male 16 (88.8%) 27 (64.3%)Disease

AML 5 (27.7%) 13 (31.0%)ALL 3 (16.7%) 5 (11.9%)CML 0 (0%) 1 (2.4%)CLL 4 (22.2%) 3 (7.1%)MM 0 (0%) 8 (19.0%)MDS 1 (5.5%) 1 (2.4%)Malignant lymphoma 5 (27.7%) 11 (26.2%)

Haematopoietic stem cell transplant

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116 patients, 40% were neutropenic, 68% had received mould-activeantifungal agents, and 36% were treated with β-lactam antibiotics atthe time of BAL sampling. Sixty-one percent (71/116) of patients hadtheir bronchoscopy performed within 3 days of the radiologicalstudies (median: 3 days; range, 0–25). Without inclusion of theresults of BAL GM and PCR, a total of 18 IPA (3 proven and 15probable), 50 possible IFDs, 6 invasive non-Aspergillus mouldinfections, and 42 no IFD cases were documented (Fig 1), yielding a15.5% prevalence rate of proven/probable IPA. Probable invasiveaspergillosis (IA) cases were diagnosed based on mycological resultsfrom cytology and microscopy (n = 10), cultures (n = 11), and/orserum GM (n = 2). The demographic features of patients in the proven/probable IPA group and controls (i.e., no IFD) were comparable (Table 1).

Allogeneic 5 (27.7%) 12 (28.6%)Autologous 2 (11.1%) 5 (11.9%)

Neutropeniaa at time of bronchoscopy 10 (55.5%) 18 (42.9%)Duration: days; median (range) 14 (7–30) 10.5 (4–30)

Mould-active antifungal agent at thetime of BAL

10 (55.5%) 23 (54.8%)

Empirical 6 (60%) 11 (47.8%)Prophylaxis 4 (40%) 12 (52.2%)

Antifungal agent usedb at thetime of BALLiposomal amphotericin B 4 (22.2%) 4 (17.4%)Voriconazole 5 (27.7%) 9 (39.1%)Posaconazole 0 (0%) 10 (43.5%)Caspofungin 1 (5.5%) 0 (0%)

P N 0.05 for all comparisons (by Mann-Whitney U-test for continuous variables andFisher's exact test for categorical variables).AML = acute myeloid leukaemia; ALL = acute lymphoblastic leukaemia; CML =chronic myeloid leukaemia; CLL = chronic lymphocytic leukaemia; MM = multiplemyeloma; MDS = myelodysplastic syndrome.

3.1. Assay performance— BAL GM

The median BAL GM OD index in the proven/probable IPA groupwas 1.11 (range, 0.095–6.90) and significantly higher than themedianin the control group (0.23; range, 0.061–1.18) (P b 0.001). Table 2summarises the performance of BAL GM testing for the diagnosis ofIPA using 3 different cut-off values for positivity. Lowering the indexcut-off from 1.0 to 0.8 improved the sensitivity without compromisingspecificity. In the no IFD control group, 3 false-positive BAL GM resultswere detected, all of which related to non-specific radiology findings.ROC analysis indicated that optimal discriminatory capability wasachieved with a BAL GM index cut-off of 0.8 (Fig 2). A 100% specificitycould be achieved at the GM index threshold of 1.65, whereasdecreasing the threshold to 0.08 would result in a 100% sensitivity.

a ANC b0.5 × 109/L.b Some patients had changes in the antifungal agent used.

3.2. Assay performance—BAL PCR

The positivity rate of PCR in BAL fluid was significantly greater inthe proven/probable IPA group as compared with the controls (78%versus 21%; P b 0.001). Performance estimates of PCR in BAL fluid areoutlined in Table 2. Nine false-positive PCR results were determined;of these, 3 were most likely attributed to airway samples contami-nated with Aspergillus spp. given the positive culture of bronchialwashings, whilst the remaining 6 cases were associated with non-specific radiology findings.

No IFD(n = 42)

Possible IFD(n = 26)

Proven/probable non-Aspergillus

IMD (n = 4)

Proven/ProbableIPA (n = 44)

Without inclusion of BAL fluid results of GM and Aspergillus PCR

No IFD(n = 42)

BAL GM (≥0.8) n = 3BAL PCR+ n = 9

Possible IFD(n = 50)

BAL GM (≥0.8) n = 10BAL PCR+ n = 18

Proven/probable non-Aspergillus

IMD (n = 6)*

BAL GM (≥0.8) n = 3BAL PCR+ n = 2

Proven/ProbableIPA (n = 18)

BAL GM (≥0.8) n = 11BAL PCR+ n = 14

Bronchoscopy/BAL (N = 116)

Inclusion of BAL fluid results of GM and Aspergillus PCR

Fig. 1. Level of certainty of diagnosis of IFD without and with the BAL GM and PCR results. Inclusion of BAL GM and PCR results increased the number of proven/probable IPA casesfrom 18 to 44, attributed to upgraded probable IPA cases from non-Aspergillus IMD group (n = 2) and possible IFD group (n = 24). Furthermore, the use of GM and PCR testsconfirmed 7 probable IPA with fungal hyphae seen on BAL fluid but no culture as due to Aspergillus. BAL PCR+ = positive results using BAL fluid with an Aspergillus PCR assay⁎Include 2 proven and 1 probable mucormycosis (Rhizopus oryzae, n = 1), 1 proven scedosporiosis (Scedosporium prolificans), 1 probable scedosporiosis (Scedosporiumapiospermum, positive bronchial washings cultures), and 1 probable Scopulariopsis disease.

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3.3. Concordance between GM and PCR in BAL fluid

For IPA, the concordance between GM and PCR from BAL fluid was79% (11 of 14 results). Three cases of IPA were detected with PCR butmissed with BAL GM (OD index b0.8), and there were no cases of IPAwith positive BAL GM but negative PCR results. For the no IFD group, a71.4% (30 of 42 results) concordance rate of negative results betweenBAL GM and PCR test was noted.

.

Table 2Diagnostic values for BAL GM using 3 different index cut-off values and BAL Aspergillus PCR for patients classified as proven/probable IPA (n = 18) versus controls (no IFD).

Assay (n/N) Performance value (% [95% CI])

Sensitivity Specificity PPV NPV PLR NLR DOR

BAL GM index cut-off≥0.5 (11/18) 61.1 (40.3–77.2) 85.7 (76.8–92.6) 64.7 (42.7–81.8) 83.7 (75.0–90.5) 4.3 (1.7–10.5) 0.45 (0.25–0.77) 9.4 (2.2–42.6)≥0.8 (11/18) 61.1 (41.3–72.9) 92.9 (84.4–97.9) 78.6 (53.1–93.8) 84.8 (77.0–89.4) 8.6 (2.6–35.1) 0.42 (0.28–0.69) 20.4 (3.8–126.9)≥1.0 (10/18) 55.6 (36.0–67.4) 92.9 (84.5–97.9) 76.9 (49.9–93.4) 83.0 (75.5–87.5) 7.8 (2.3–32.8) 0.48 (0.33–0.76) 16.3 (3.1–98.6)

BAL PCR (14/18) 77.8 (56.3–91.9) 78.6 (69.4–84.6) 60.9 (44.0–71.9) 89.2 (78.7–96.0) 3.6 (1.8–6.0) 0.28 (0.10–0.63) 12.8 (2.9–62.1)

PLR = positive likelihood ratio; NLR = negative likelihood ratio; DOR = diagnostic odds ratio.

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3.4. Impact of using BAL GM and PCR as adjunct diagnostic results

If GM and PCR testing of BAL specimens were used as an adjunctfor diagnosis, 7 patients diagnosed as probable IPA based on positivemicroscopy (fungal hyphae) and characteristic radiology featureswould have been confirmed as Aspergillus infections, giving moreconfidence that the clinicians were indeed treating IPA. Twenty-fourpatients diagnosed as possible IFD, based on characteristic radiologyfindings only, would have been upgraded to probable IPA (Fig 1).

3.5. Impact of mould-active antifungal agent, neutropenia, andpiperacillin-tazobactam

Ten of the 18 patients with proven/probable IPA had receivedmould-active antifungal agent for a median of 19 days (range, 6–77)prior to the bronchoscopy. The sensitivity of BAL GM in these patientsreceiving mould-active antifungal agents before BAL sampling wasnot different to those not on antifungal agents (50 versus 75%). Noreduction in the mean sensitivity of PCR in BAL was observed (70% inthe subset receiving antifungal agents versus 88% in those not onantifungal agents).

Among patients with proven/probable IPA, the sensitivity of GMtesting in BALwas 40% in thosewith neutropenia (n = 10) and 88% inthose who were not neutropenic (n = 8). The sensitivity of PCR inBAL was 60% in neutropenic and 100% in non-neutropenic proven/probable IPA patients (P = 0.092). The use of mould-active antifungalagents prior to bronchoscopy was greater among neutropenic (8/10,80%) versus non-neutropenic (2/8, 25%) patients with proven/probable IPA.

Fig. 2. ROC curve for BAL GM without its inclusion for case definition (area under thecurve = 0.790, SE = 0.076, 95% CI = 0.641–0.940).

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Approximately 38% (16/42) of patients in the control group weretreated with β-lactam antibiotics, predominantly piperacillin-tazobactam, at the time of bronchoscopy. Of these, 2 (13%) hadfalse-positive GM results.

4. Discussion

The diagnostic accuracy of Aspergillus GM-ELISA and PCR in BALhas been the focus of many studies. Earlier reports have limitedgeneralizability to routine clinical practice as some have excludedpatients who had received either mould-active antifungal agents,β-lactam antibiotics, or restricted duration of antifungal agents to≤2 days prior to bronchoscopy (Maertens et al., 2009; Racil et al.,2011). Additionally, many studies were subject to pitfalls in studydesign such as incorporation bias or lack of blinding (Frealle et al.,2009; Hsu et al., 2010; Luong et al., 2010; Park et al., 2010; Penacket al., 2008; Racil et al., 2011; Sanguinetti et al., 2003). Whilst thesediagnostic tests may have excellent sensitivity and specificity incontrolled settings, the clinical utility of GM and PCR testing of BALfluid in routine everyday clinical practice has not been wellexamined, particularly from a multicentre experience.

In the present study, GM testing in BAL was found to havemoderate sensitivity (61%) at the OD index of 0.8. This sensitivity wasconsiderably lower than that found in a number of previous studies(90–100%) (Becker et al., 2003; Hsu et al., 2010; Luong et al., 2010;Maertens et al., 2009; Penack et al., 2008) but similar to those studiesthat included a large number of patients receiving antifungal agents(57–67%) (Bergeron et al., 2010; Musher et al., 2004; Nguyen et al.,2011; Racil et al., 2011; Reinwald et al., 2012b). The specificity (93%)and negative likelihood ratio (0.42) of BAL GM were excellent. Thefalse-positive BAL GM results in patients with non-Aspergillus mouldinfections may reflect undetected co-infection or airway colonisationwith Aspergillus or cross-reaction with β-lactam antibiotics. An ODindex of 0.8, which resulted in the best overall performance estimates,was determined to be the most appropriate cut-off for positivemycological evidence in our study. This index cut-off is consistentwith several earlier observations (0.8–1.0) (Becker et al., 2003;D'Haese et al., 2012; Frealle et al., 2009; Maertens et al., 2009; Nguyenet al., 2011) but higher than that proposed by others (0.5) (Bergeronet al., 2010; Musher et al., 2004; Park et al., 2010; Penack et al., 2008;Racil et al., 2011).

We found that the PCR assay was more sensitive than GM test;however, this was offset by a decrease in specificity. Diagnostic oddsratio, which indicates the overall diagnostic accuracy of a test,favoured the BAL GM test over PCR. More false-positive results wereseen with the PCR assay, possibly reflecting the limited discriminatorypower for colonisation versus invasive disease (Tuon, 2007) or earlyinfection. Despite some differences in PCR methodology, we notedthat the sensitivity of PCR in BAL in the present study was similar tothat of an earlier meta-analysis (sensitivity: 79%; specificity: 94%)(Tuon, 2007) and in clinical studies involving haematology patients(sensitivity: 58–77%; specificity: 87–100%) (Khot et al., 2008; Musheret al., 2004; Reinwald et al., 2012a; Reinwald et al., 2012b).

s galactomannan and PCR in bronchoalveolar lavage fluid for the4), http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.020

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In our cohort, no improvement in sensitivity was observed withthe combination of GM and PCR due to the greater sensitivity of PCR;however, specificity would be increased to 100% if negativity wasdefined by negative result of either test. This finding is consistent withthe results of a meta-analysis focusing solely on the hematologypopulation, which found only a marginal increase (~6%) in the overallsensitivity with combined BAL GM and PCR testing (Heng et al., 2013).If resources are limited, then either BAL GM or PCR in conjunctionwith radiology appears to be feasible. A combination of both tests withradiology may have the potential to optimize sensitivity andspecificity, but this needs to be determined in a larger study.

Both BAL GM and PCR tests allowed clinicians to differentiatebetween IPA and probable IFD (fungal hyphae on microscopy butnegative culture) and to upgrade possible IA (based on radiologychanges only) to probable IPA. This is a critically important utility ofBAL GM and PCR as it allows clinicians to direct antifungal therapywith confidence, particularly in those receiving mould-active anti-fungal prophylaxis where non-Aspergillus mould breakthroughinfections have to be considered.

The test sensitivity of BAL GM and PCR was not found to bedecreased by the use of mould-active agents in the present study,although the effect could be significant if the study population waslarger. We did not observe any increase in the test sensitivity withneutropenia. Therapeutic bias may play a role in this circumstance orpossibly a sample size effect. In the current study, β-lactam antibioticappeared to exert a small effect on the specificity of BAL GM, althougha recent study conducted during October 2009–2010 suggested thatthe association between piperacillin-tazobactam preparation andfalse-positive GM results is diminishing (Mikulska et al., 2012).

This study has several limitations, which may have affected thediagnostic yield of GM and PCR in BAL. Firstly, the use of the EORTC/MSG consensus definitions as the reference standard may havecontributed to the apparent lower test performance. Failure to meetthe consensus definition does not absolutely exclude the disease butmerely indicates insufficient evidence to establish the diagnosis(Donnelly, 2010). Misclassification of cases would therefore under-estimate the tests' sensitivity and specificity. Second is the relativelysmall number of case patients with proven or probable IPA. However,as the incidence of IA in the haematology population has substantiallydecreased due to widespread use of mould-active antifungal prophy-laxis (Ananda-Rajah et al., 2012), our results reflect real-world currentpractice. Third, the variable amount of saline that may have beeninstilled and fluid recovered during BAL procedure, inter-individualand across different centres, may affect GM and DNA concentrationand subsequently the test sensitivity and cut-off value for BAL GM.However, 1 study has found that the volume of solution instilled hadno significant effect on the positivity of BAL GM test (P = 0.171), butthere was a trend towards negative BAL GM results with highervolume of aspirated fluid (P = 0.092) (Racil et al., 2011).

In conclusion, this study of unselected haematology patientsundergoing bronchoscopy showed that BAL GM at an OD index cut-offof ≥0.8 was of moderate sensitivity for the diagnosis of IPA but highlyspecific. Aspergillus PCR in BAL fluid had comparatively greatersensitivity, yet the specificity was lower. The major clinical utility ofGMand PCR in BALwould be to rule out a diagnosis of IPA if resultswerenegative. Combining either test with radiology would maximise thesensitivity and specificity and add certainty to the diagnosis of IPAallowing cases identified as IFD cases to be reclassified as due toAspergillus spp. The use and cost-effectiveness of combining both testswith radiologymay require further investigation in larger clinical study.

Acknowledgments

MS and SCAC have sat on advisory boards for and receivedresearch funding (not related to the current work) from Pfizer, MSD,and Gilead Sciences. COM has been a member of advisory boards for,

Please cite this article as: Heng S-C, et al, Clinical utility of Aspergilludiagnosis of invasive pulmonary aspe..., Diagn Microbiol Infect Dis (201

received investigator-initiated grants from (not related to the currentwork), and given lectures for Gilead Sciences, Pfizer, MSD, and OrphanAustralia. DCMK has sat on an advisory board for Pfizer and receivesfinancial support (not related to the current work) from Pfizer, Roche,MSD, Novartis, and Gilead Sciences. All other authors: no conflict ofinterest. Preliminary data from this work were presented at the 53rdInterscience Conference on Antimicrobial Agents and Chemotherapy,Denver, CO, USA, 2013, abstract M-1273.

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