prospective assessment of platelia™aspergillus galactomannan antigen for the diagnosis of invasive...

American Journal of Transplantation 2004; 4: 796–802Blackwell Munksgaard

Copyright C© Blackwell Munksgaard 2004

doi: 10.1111/j.1600-6143.2004.00415.x

Prospective Assessment of PlateliaTM AspergillusGalactomannan Antigen for the Diagnosis of InvasiveAspergillosis in Lung Transplant Recipients

Shahid Husaina, Eun Jeong Kwaka, Asia

Obmanb, Marilyn M. Wagenera, Shimon Kusnea,

Janet E. Stoutb, Kenneth R. McCurrya

and Nina Singha,b,∗

aUniversity of Pittsburgh Medical Center, Thomas E.Starzl Transplantation Institute, and bVeterans AffairsMedical Center, Pittsburgh, PA∗Corresponding author: Nina Singh, [email protected]

The clinical utility of PlateliaTM Aspergillus galac-tomannan antigen for the early diagnosis of invasiveaspergillosis was prospectively assessed in 70 consec-utive lung transplant recipients. Sera were collectedtwice weekly and tested for galactomannan. Invasiveaspergillosis was documented in 17.1% (12/70) of thepatients. Using the generalized estimating equationmodel, at the cutoff value of ≥≥ 0.5, the sensitivity ofthe test was 30%, specificity 93% with positive andnegative likelihood ratios of 4.2 and 0.75, respectively.Increasing the cutoff value to ≥≥ 0.66 yielded a sensitiv-ity of 30%, specificity of 95%, and positive and negativelikelihood ratios of 5.5 and 0.74. A total of 14 patientshad false-positive tests, including nine who had cys-tic fibrosis or chronic obstructive pulmonary disease.False-positive tests occurred within 3 days of trans-plantation in 43% (6/14) of the patients, and within 7days in 64% (9/14). Thus, the test demonstrated excel-lent specificity, but a low sensitivity for the diagnosis ofaspergillosis in this patient population. Patients withcystic fibrosis or chronic obstructive pulmonary dis-ease may transiently have a positive test in the earlypost-transplant period.

Key words: Aspergillus, fungal infections, lung trans-plants

Received 24 October 2003, revised and accepted forpublication 2 January 2004

Introduction

Invasive aspergillosis has long been recognized as amongthe most significant opportunistic infections of lung trans-plant recipients. Aspergillus infections have been re-ported in an average 6–8% and up to 16% of lung

transplant recipients (1–5). Impairment of local host de-fenses, e.g. mucociliary clearance, ischemic airway in-jury, altered alveolar phagocytic function, direct commu-nication of the transplanted organ with the environmentand an overall higher intensity of immunosuppression,render these patients uniquely susceptible to invasiveaspergillosis (2,3). Indeed, patients undergoing lung trans-plantation are more likely than other organ transplant re-cipients to develop invasive aspergillosis. Mortality in lungtransplant recipients with invasive aspergillosis rangesfrom 60 to 74%; an estimated 9% of deaths followinglung transplantation are considered attributable to invasiveaspergillosis (3).

Failure to establish an early diagnosis remains a majorimpediment to the effective management of invasive as-pergillosis. Culturing is an insensitive means of diagnosingAspergillus infections and fails to detect 30–50% of inva-sive aspergillosis cases. While high-resolution tomographicimaging studies to detect halo and air-crescent signs haveproven useful for early diagnosis of invasive aspergillosis inneutropenic patients, these signs are distinctly unusual insolid organ transplant recipients with invasive aspergillosis(3). Indeed, Aspergillus infections in lung transplant recip-ients frequently lack a characteristic radiographic appear-ance and present most often as focal areas of patchy con-solidation (5).

In recent years, efforts have been directed towards identi-fying noninvasive diagnostic markers for the rapid and reli-able detection of invasive aspergillosis. One such marker,galactomannan, is a polysaccharide cell wall componentof Aspergillus, which is released in variable quantities inthe serum during fungal growth in the tissues. Each galac-tomannan has up to 10 epitopes, making it possible for theantibody to be used both as captor and as detector (6). Thesandwich-enzyme immunoassay (EIA) for the detection ofgalactomannan has been evaluated in several immuno-compromized patient populations with varying sensitivityand specificity (7–11). To date, however, the role of galac-tomannan antigen for the diagnosis of invasive aspergillo-sis has not been assessed in lung transplant recipients. Thegoals of this study were to evaluate the utility of Bio-RadLaboratories PlateliaTM Aspergillus EIA (Redmond, WA) forearly diagnosis of invasive aspergillosis in lung transplantrecipients.

796

Galactomannan for the Diagnosis of Invasive Aspergillosis in Lung Transplantation

Methods

Patients

Seventy consecutive patients undergoing lung transplantation at the Univer-sity of Pittsburgh Medical Center were prospectively enrolled between June2001 and December 2002. Blood samples were collected twice weekly(Tuesdays and Thursdays) during the post-transplant hospital stay and sub-sequent hospitalizations. All patients were followed prospectively for upto 18 months after transplantation. The institutional review board approvedthe study and informed consent was obtained before study entry from eachpatient.

Immunosuppressive regimen

The immunosuppressive regimen comprised of tacrolimus (with targettrough levels of 10–12 ng/mL), azathioprine (2 mg/kg per day) and pred-nisone. In addition these patients received daclizumab induction therapy (1mg/kg) intravenously on days 0, 14, 28, 42 and 56. Mycophenolate mofetiland sirolimus were substituted for azathioprine and tacrolimus, respectively,in selected cases with recurrent acute rejection and chronic allograft rejec-tion. Patients transplanted after May 2002 received preoperative inductionwith rabbit antithymocyte globulin (Thymoglobulin; Sangstat, Menlo Park,CA), as previously reported (12). Their maintenance immunosuppressiveregimen consisted of tacrolimus with a target trough concentration of 5–8ng/mL, and low-dose prednisone. Acute rejection episodes were treatedwith methylprednisolone 1 g for three consecutive days or an oral pred-nisone taper. Muromonab-CD3 (Orthoclone OKT3, Ortho Biotech, Raritan,NJ) was used for corticosteroid-resistant rejection episodes.

Antimicrobial prophylaxis

In patients transplanted from June–November 2002, cytomegalovirus(CMV) pp65 antigenemia-directed preemptive therapy with ganciclovir wasemployed as CMV prophylaxis. Patients at low risk for CMV disease (CMV-seropositive recipients, regardless of the donor CMV serologic status, orCMV-seronegative recipients receiving an organ from a CMV -seronegativedonor) received IV ganciclovir 5 mg/kg q12 if weekly surveillance for pp65antigenemia showed ≥ 10 CMV positive cells/2 × 105 leukocytes. Pa-tients at higher risk for CMV disease (CMV-seronegative recipients of CMVseropositive donors) received IV ganciclovir 5 mg/kg q12 if weekly surveil-lance for pp65 antigenemia showed ≥ 1CMV positive cells 2 × 105 leuko-cytes. IV ganclovir was continued until two consecutive CMV antigenemiatests were negative. Starting December 2002, all patients received oral val-ganciclovir 450 mg twice daily as CMV prophylaxis. The dosage of valganci-clovir was adjusted for renal dysfunction. Valganciclovir was continued for 6months in the high-risk patients and for 3 months in the low-risk individuals.

A majority of the patients received antifungal prophylaxis with 200 mg oforal fluconazole daily for 3 months. In all patients with pre or post-transplantcolonization with Aspergillus species, and in some patients with cytic fibro-sis, however, fluconazole was substituted with oral itraconazole 200 mgtwice daily, with or without inhalational amphotericin B deoxycholate. Fif-teen patients received 200 mg bid of oral voriconazole for 3 months aftertransplantation as antifungal prophylaxis.

Definitions

Invasive aspergillosis was diagnosed prospectively and categorized intoproven and probable groups as per the EORTC/MSG criteria (13) by theinvestigators who were blinded to the galactomannan antigen testing re-sults. Proven invasive aspergillosis was defined as isolation of Aspergillusspecies from a normally sterile body fluid or histopathologic or cytopatho-logic examination showing hyphae from biopsy specimen with the evidenceof associated tissue damage (13). Probable invasive aspergillosis was de-fined as presence of both mycological and clinical criteria in lung transplants.A mycological criterion was fulfilled with the isolation of Aspergillus species

from bronchoalveolar lavage samples, and the clinical criterion was satis-fied by the presence of either one major or two minor criteria as proposedby the European Organization for the Research and Treatment of Cancerand the Mycoses Study Group (13). Development of a new infiltrate wasconsidered as a clinical criterion only when the presence of bacterial and/orviral infections was ruled out by culture and rejection was excluded bytissue biopsy. Aspergillus tracheobronchitis was defined as isolation of As-pergillus in culture from ulcerative necrotic lesions in the tracheobronchialtree or the bronchial anastomosis, and histopathologic evidence of tissueinvasion (5,14). As all cases of trancheobronchitis were histopathologicallydocumented, they were considered proven invasive aspergillosis. The re-sults of the test were not used in the management of the patients andclinical care providers were blinded to the results.

Galactomannan antigen testing

Serum samples were stored at − 80 ◦C within 24 h of collection. The As-pergillus galactomannan antigen was detected by one-stage immunoen-zymatic sandwich microplate assay (Bio-Rad Laboratories PlateliaTM As-pergillus EIA). Samples were processed as per manufacturer’s instructions.Briefly, 300 lL of the test serum was added to 100 lL of 4% EDTA solution.After vigorous homogenization, the tubes were heated at 100 ◦C in a waterbath for 3 min, followed by centrifugation of the tube at 10 000 g for 10 min.Fifty microliters of the supernatant and 50 lL of the horseradish peroxidase-labeled monoclonal antibody (EBA-2) were incubated in the EBA-2 coatedmicroplates for 90 min at 37 ◦C. After five washes, the plates were incu-bated with 200 lL of substrate chromogen reaction solution for 30 ± 5 minin the dark with the temperature ranging from 18 to 25 ◦C. The reaction wasstopped with 1.5 N sulfuric acid solution. Optical density (OD) was read at450 nm with a reference filter of 620 nm. Positive, negative, and cut-offcontrols were incorporated in each assay. An OD index of 0.5 was consid-ered positive. All positive samples were retested and considered positiveonly if the repeat test was also positive.

Statistical analysis

Sensitivity and specificity were calculated in reference to the diagnosis ofinvasive aspergillosis by using the total number of patients in the study.Only those tests performed within a week of the diagnosis of invasiveaspergillosis were considered for this analysis. A 1-week limit was chosenso as to avoid the inclusion of tests that could falsely increase the sensitivity.The rationale for using this criterion was that a positive test after 1 weekof the diagnosis might be clinically irrelevant and have little implications forpatient management. Specificity was also determined based on the totalnumber of tests in the patients without evidence of invasive aspergillosis.

As all observations within a patient (galactomannan tests in context of thisstudy) are correlated, we used the generalized estimating equation (GEE)model to account for the correlation in multiple observations from the samesubject (15). The binary outcome was analyzed using a clustered logisticmodel with robust variance to assess the sensitivity and specificity. Receiveroperative curves (ROCs) were generated to determine the optimal cutoffpoint of the test. In the GEE model, only those tests that were performedwithin a week of the gold standard (biopsy and culture) were used. Thenumber of tests performed per patient and the days post transplant to onsetof infection were compared using the Mann–Whitney test. Categorical data(use of antifungal agents, prior history of rejection, etc.) were comparedusing the Chi-square or Fisher Exact test.

Results

Demographics and clinical characteristics of the 70 studypatients are outlined in Table 1. The most commonindications for transplantation were chronic obstructive

American Journal of Transplantation 2004; 4: 796–802 797

Husain et al.

Table 1: Demographics and clinical characteristics of the studypatients

Age, yearsMedian (range) 54 (21–68)

SexMale 47.1% (33/70)Female 52.9% (37/70)

Type of transplantSingle lung 52.9% (37/70)Double lung 45.7% (32/70)Heart-lung 1.4% (1/70)

Underlying lung diseaseChronic obstructive pulmonary disease 40% (28/70)Cystic fibrosis 15.7% (11/70)Idiopathic pulmonary fibrosis 15.7% (11/70)Primary pulmonary hypertension 7.7% (5/70)Alpha-one antitrypsin deficiency 7.1% (5/70)Silicosis 4.2% (3/70)Sarcoidosis 4.2% (3/70)Rheumatoid lung disease 4.2% (3/70)Bronchiectasis 1.4% (1/70)

Immunosuppressive agent∗Tacrolimus 90% (63/70)Cyclosporine A 2.8% (2/70)Mycophenolate mofetil 25.7% (18/70)Sirolimus 8.6% (6/70)Azathioprine 50% (35/70)

∗Some patients received more than one agent

pulmonary disease [COPD (40%)], followed by cystic fi-brosis (16%) and idiopathic pulmonary fibrosis (16%).The overall incidence of invasive aspergillosis was 17.1%(12/70), including nine patients with proven and three withprobable invasive aspergillosis. Fifty-nine percent (7/12) ofthe patients had pulmonary aspergillosis, 33% (4/12) hadAspergillus tracheobronchitis and one patient had systemicinvasive aspergillosis. Median time to onset was 31 days(range 7–407 days). Aspergillus fumigatus was the mostcommon isolate (75%, 9/12) followed by Aspergillus flavus,Aspergillus niger and Aspergillus versicolor in one caseeach.

Performance characteristics of the test

A total of 891 sera were analyzed from 70 patients. Themedian number of tests performed was comparable forpatients with and without invasive aspergillosis (13.5 vs.8, p > 0.5). The galactomannan test was positive in threeof 12 patients with invasive aspergillosis. The positive testpreceded the histological diagnosis by 3 days in a patientwith pulmonary aspergillosis. The test was positive 4 daysafter the histological diagnosis in a patient with systemicinvasive aspergillosis, and 7 days following the diagnosisof proven pulmonary aspergillosis in the third patient. Twoof three patients with the positive test who did not receiveantifungal prophylaxis had multiple positive tests within aweek of the diagnosis (mean number of positive tests 2.5).The third patient, who had received prophylaxis with itra-conazole for 14 days before the diagnosis, had a single pos-itive test (Table 2).Two patients (one probable pulmonary T

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798 American Journal of Transplantation 2004; 4: 796–802


Table 3: Sensitivity of galactomannan test according to clinicalsyndromes of invasive aspergillosis

Systemic aspergillosis 100% (1/1)Aspergillus tracheobronchitis 0% (0/4)Pulmonary aspergillosis 28.5% (2/7)Pulmonary and systemic aspergillosis 37.5% (3/8)

and one tracheobronchitis) did not have galactomonnan as-say drawn 1 week before the diagnosis.

Nine of the 12 patients with invasive aspergillosis did nothave a positive test within a week of the diagnosis. Whenanalyzed according to the number of patients, the overallsensitivity and specificity was 25% (3/12) and 76% (44/58),respectively. The sensitivity was 22% (2/9) in cases ofproven invasive aspergillosis and 33% (1/3) in those withprobable invasive aspergillosis. The sensitivity and speci-ficity excluding tracheobronchitis were 37.5% (3/8) and75.8% (44/58), respectively (Table 3). A total of 891 serumsamples collected from 70 lung transplant recipients in-cluded 133 sera from nine patients with proven invasiveaspergillosis, 68 sera from three patients with probable in-vasive aspergillosis, and 698 sera from 58 patients withoutevidence of invasive aspergillosis. Based on the number oftests, the specificity of the test was 95% (654/690).

We calculated the sensitivity and specificity and the like-lihood ratios using the GEE regression analysis. For themodel, 306 samples drawn within a week of the perfor-mance of gold standard (biopsy and culture) were analyzed.Based on this model, at a cutoff value of 0.5, the sensitiv-ity was 30%, and specificity was 93%, with positive andnegative likelihood ratios of 4.2 and 0.75, respectively. In-creasing the cutoff value to 0.66 yielded a sensitivity of30%, specificity of 95%, and positive and negative likeli-hood ratios of 5.5 and 0.74 (Figure 1). However, increasingthe cutoff to 1 decreased the sensitivity to 20%.

Nine patients with invasive aspergillosis who had false-negative tests included five patients with pulmonary as-pergillosis (two proven, and three probable) and four pa-tients with Aspergillus tracheobronchitis. Only two of fivepatients with pulmonary aspergillosis and false-negativetests had received antifungal prophylaxis before thehistopathological diagnosis (amphotericin B for 2 weeksin one patient, and itraconazole that was later substitutedwith a lipid formulation of amphotericin B for 4 weeks inanother patient). Of four patients with tracheobronchitis,two had received antifungal prophylaxis: one with a lipidformulation of amphotericin B, and one with itraconazole.The number of tests performed per patient (median 12 vs.15) and the time of onset of invasive aspergillosis post-transplant (median 38 vs. 18 days) did not differ signifi-cantly for patients with false-negative as compared withthose with true-positive tests.

Fourteen patients without invasive aspergillosis had 36false-positive test results. The false-positive test occurred

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False-Positive Rate0.00 0.25 0.50 0.75 1.00

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Figure 1: Receiver operative curve (ROC) characteristics in the

generalized estimation equation (GEE) model for the galac-

tomannan test. The ROC is plotted between the true-positive rate(sensitivity) on the y -axis, and the false-positive rate (1-specificity)on the x-axis. An ideal ROC cure is one that arches up to the upperleft-hand corner of the graph. Area under curve (AUC) representsthe accuracy of the galactomannan test and was 0.634 (SE, 0.118;95% CI, 0.40–0.86).

within 3 days of transplantation in 43% (6/14) of the pa-tients, within 7 days in 64% (9/14), and within 14 daysof transplantation in 79% (11/14) of these patients. Themedian galactomannan index value in the patients withfalse-positive tests was 0.868. None of these patientswas receiving piperacillin-tazobactam at the time of thefalse-positive tests. Colonization with Aspergillus (6/14 vs.16/44), use of prior antifungal prophylaxis (8/14 vs. 16/44),and prior rejection episodes (6/14 vs. 20/44) did not differsignificantly for patients with false-positive tests as com-pared with those with true-negative tests. Similarly no cor-relation was noted between the use of various immuno-suppressive drugs and performance characteristics of thetest.

Of 14 patients with false-positive tests, five had consec-utive multiple positive tests. The median number of posi-tive tests in these patients was 3 (range 2–9) with an indexvalue of 0.9. Three of the five patients had cystic fibrosisas their underlying diagnosis and had received voricona-zole or an intravenous lipid formulation of amphotericinB or itraconazole as antifungal prophylaxis. All were col-onized with mucoid P. aeruginosa. However, colonizationwith Aspergillus species was not documented in these pa-tients. The fourth patient had COPD, did not receive anti-fungal prophylaxis, and had methicillin-resistant S. aureusbacteremia. The last lung transplant recipient with multi-ple false-positive tests had bronchiectasis (owing to graftvs. host disease following sibling-matched bone marrowtransplantation 6 years earlier), and had not received anti-fungal prophylaxis.

Mortality

One-year mortality in the entire cohort was 14.2% (10/70).Autopsy was performed on all patients who died during


Husain et al.

the course of study. Of patients with invasive aspergillo-sis, 3/12 died. These included two patients with pulmonaryinvasive aspergillosis and one patient with systemic inva-sive aspergillosis. None of the patients with Aspergillus tra-cheobronchitis died. Of seven patients who died of othercauses, none had invasive aspergillosis on autopsy.

Discussion

Studies evaluating the role of galactomannan assay for theearly diagnosis of invasive aspergillosis have largely beenconducted in patients undergoing cancer chemotherapy orhematopoietic stem cell transplantation. Serial monitoringof the test in these patients has documented a sensitivityof 67–93% and specificity of 86–95% for the diagnosis ofinvasive aspergillosis (16–18). Others have reported lowersensitivity in the same patient population (9). In patientswithout hematological malignancy or hematopoietic stemcell transplantation, the galactomannan assay has shownan excellent specificity, but a consistently lower sensitivity.In nine liver transplant recipients with invasive aspergillosisand 33 control patients without invasive aspergillosis afterliver transplantation, the sensitivity of the test was 55.6%and the specificity was 93.9% (19). Likewise, in 52 predom-inantly nonimmunocompromized patients with invasive as-pergillosis, the sensitivity was 38% and the specificity was100% (11). Using the GEE model, the galactomannan testdemonstrated a specificity of 95%, but a relatively low sen-sitivity (30%) for the diagnosis of invasive aspergillosis inour lung transplant recipients.

A number of factors could have accounted for the lowersensitivity observed in our patients. The method of calcu-lation may influence the performance characteristics of thetest. Previous studies in patients with invasive aspergillo-sis have used different denominators for the calculationof sensitivity and specificity (8). Ideally, either the num-ber of tests or the patients should be used to calculateboth values. Use of patients in the denominator typicallyreduces specificity, while the use of the number of testresults in falsely high specificity owing to the inclusion of alarge number of negative tests. The GEE regression modelused in our study obviates the aforementioned biasesand results in a more conservative value approaching themean. This model is particularly well suited for analyzingoutcome where the variables are correlated and repeatedcontinually.

Lower galactomannan index values have been noted inanimal models of invasive aspergillosis treated with anti-fungal agents, e.g. amphotericin B (20,21), voriconazole(22) and posaconazole (23), and in patients receiving can-cer chemotherapy or undergoing hematopoietic stem celltransplantation (8,24). The use of antifungals may lowergalactomannan values by decreasing the fungal load (24).The sensitivity of the test was only 20% among five neu-tropenic patients receiving antifungal prophylaxis (17). The

use of antifungal prophylaxis might have led to a lower sen-sitivity of the test in our patients. We, however, note thatthe employment of antifungal prophylaxis in these high-risk patients is a common practice (25,26). Indeed, a recentsurvey documented that 76% of the U.S. lung transplantcenters employ antifungal prophylaxis in all patients, and24% use it in selected patients for a median of 3 monthsafter transplantation (25). In this context, our results depictthe diagnostic accuracy and the performance of this testin the actual clinical setting in which it will be used.

Another factor that may have contributed to a lower sensi-tivity is the absence of neutropenia in our patients. The sen-sitivity of the test was 15.7% in patients with chronic gran-ulomatous disease, 16.7% in those with Job’s syndrome,and 80% in other immunocompromized patients with neu-tropenia (p < 0.0002; 27). In a non-neutropenic patient withchronic granulomatous disease and invasive aspergillosis,the test was positive in the abscess, but the galactoman-nan antigen was undetectable in the serum (10). A lowersensitivity of the test in non-neutropenic patients may berelated to a lower fungal burden, as the ability to clearthe fungal mannan from the bloodstream by macrophagemannosyl receptors remains unimpaired in patients with-out granulocytopenia (27,28).

The galactomannan test was negative in our patientswith Aspergillus tracheobronchitis and anastomotic in-fections. Characterized by endobronchial lesions rangingfrom ulcers and pseudomembranes, tracheobronchitis orbronchial anastomotic infections are a distinct form of inva-sive aspergillosis observed almost exclusively in lung trans-plant recipients. A negative test in these patients is biolog-ically plausible given that Aspergillus tracheobronchitis isa locally invasive infection and dissemination is unlikely inits early stages (5,14). That the devascularized anastomoticsite may not result in a sufficiently high level of galactoman-nan in the bloodstream to allow its detection is also plau-sible. It is possible that the use of bronchoalveolar lavagesamples for galactomannan antigen testing may be moreuseful in this patient population.

False-positive tests have been documented in 8–10% ofpatients with cancer and hematopoietic stem cell trans-plant recipients (6,16,17,29). Transient antigenemia, in-duction of antigenemia by immunosuppressive therapy orcrossreactivity with unidentified serum components havebeen proposed to account for the false-positive resultsobserved in these patients (6,30,31). Fourteen lung trans-plant recipients had a false-positive test in our study. Forty-three percent of the false-positive tests occurred within3 days, and 64% within 7 days of transplantation. Althoughgalactomannan may have crossreactivity with other fun-gal antigens, e.g. in Penicillium, Paecilomyces, and Al-ternaria, none of our patients had infections owing to thesefungi (32). Additionally, none of our patients was receivingpiperacillin-tazobactam, which may yield a positive test asrecently reported (33,34). Only 2/14 of the patients were



colonized with Aspergillus before the false-positive test.At least one other study has also shown that colonizationwith Aspergillus did not affect the specificity of the test.In nonimmunocompromized patients with pulmonary dis-ease, none of the eight patients with Aspergillus coloniza-tion had a positive test (11).

Five of 14 patients in whom the test was repeatedly pos-itive are of particular interest. Three of these five patientshad cystic fibrosis and one had COPD as underlying lungdisease. Although colonization with Aspergillus was neverdocumented in these patients, both cystic fibrosis andCOPD are conditions known to predispose to airway col-onization with Aspergillus. Whether surgery could haveresulted in translocation and transient release of galac-tomannan antigen from subclinical or undetected colo-nization remains speculative. A more likely plausibility,however, is that these patients in fact had a true-positivetest. As three of the five patients with multiple positivetests had received prophylaxis with a mould-active drug,the antifungal agent may have aborted the progression ofsubclinical infection to invasive aspergillosis.

In patients with hematologic malignancy and hematopoi-etic stem cell transplant recipients, the galactomannan testwhen serially monitored has been shown to precede thediagnosis of invasive aspergillosis by 12–30 days (31,35).Galactomannan antigen was detectable in 65% of thecases before the first clinical sign, in 71% before any lesionwas apparent radiographically, and in 44% before fever oc-curred in hematological patients (16). In another report incancer patients, however, only three of 153 episodes ofinvasive aspergillosis were first diagnosed based on thegalactomannan test (9). In our study only one patient hada positive test before the diagnosis of invasive aspergillo-sis where antigenemia preceded the histological diagno-sis of pulmonary aspergillosis by 3 days. The lack of earlypositivity of the test in our study may represent a rapidprogression of disease in these patients (9).

Studies evaluating the role of galactomannan in the diagno-sis of invasive aspergillosis have mostly used a cutoff valueof 1.0 (7,11,16,18,19,24,29). The manufacturer’s currentlyrecommended cutoff value in the USA is 0.5. On the ba-sis of our data, increasing the cutoff to 0.66 may be moreclinically relevant in lung transplant recipients, as it yieldeda higher specificity without compromising the sensitivityof the test. This cutoff also increased the positive likeli-hood ratio to 5.5 as compared with 4.4 with the standardcutoff.

How then can the test be utilized for the diagnosis ofinvasive aspergillosis in lung transplant recipients? Serialmonitoring of the galactomannan test performed routinely,as screening may have a limited role for an earlier diag-nosis of invasive aspergillosis in this patient population.Positive GM test results in this setting will increase thepost-test probability to 0.49 from 0.15, while a negative

will decrease the post-test probability to 0.11. A nega-tive test, however, in these patients may not rule outinvasive aspergillosis and a tissue diagnosis must be pur-sued. Alternate specimen, like bronchoalveolar lavage in-stead of serum samples, may prove to be advantageousin this population of lung transplant recipients who are notneutropenic.

Acknowledgment

The authors thank Bio-Rad Laboratories, Redmond, WA, for providing kitsand financial support for the study.

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