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JCM Accepts, published online ahead of print on 11 April 2007
J. Clin. Microbiol. doi:10.1128/JCM.00077-07
Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Bronchoalveolar lavage galactomannan in the diagnosis of invasive pulmonary aspergillosis among solid organ transplant recipients. Cornelius J. Clancy1,2*, Reia A. Jaber1, Helen L. Leather1, John R. Wingard1, Benjamin Staley3, L. Joseph Wheat4, Christina L. Cline1, Kenneth H. Rand1, Denise 1Department of Medicine, University of Florida College of Medicine, Gainesville, FL; 2North Florida/South Georgia Veterans Health System, Gainesville, FL; 3Shands Teaching Hospital Department of Pharmacy, Gainesville, FL; 4MiraVista Diagnostics, ACCEPTED
University of Florida College of Medicine Box 100277 JHMHC Gainesville, FL 32610 Phone: 352-379-4027 Fax: 352-379-4015 e-mail: [email protected] Running title: BAL galactomannan and solid organ transplant Key words: Aspergillus; galactomannan; bronchoalveolar lavage; solid organ transplant; diagnosis Abstract.
We review the experience at our institution with galactomannan (GM) testing of bronchoalveolar lavage (BAL) fluid in the diagnosis of invasive pulmonary aspergillosis (IPA) among solid organ transplant recipients. Among 81 patients for whom BAL GM was ordered (heart, n=24; kidney, n=22; liver, n=19; lung, n=16), there were five cases of proven or probable IPA. All five patients had BAL GM 2.1 and survived following antifungal therapy. The sensitivity, specificity, positive and negative predictive values for BAL GM at a cut-off 1.0 were 100%, 90.8%, 41.7% and 100%, respectively. The sensitivity of BAL GM was better than conventional tests such as serum GM or BAL cytology and culture. Moreover, a positive BAL GM diagnosed IPA several days to four weeks before other methods in three patients. Twelve patients had BAL GM 0.5 but no evidence of IPA. Among these, lung transplant recipients accounted for 41.7% (5/12) of the false positive results, reflecting frequent colonization of airways in this population. Excluding lung transplants, the specificity and positive predictive value for other solid organ transplants increased to 92.9% and 62.5%, respectively (cut-off 1.0). In ACCEPTED
conclusion, BAL GM facilitated more rapid diagnoses of IPA and institution of antifungal therapy among non-lung solid organ transplant recipients, and helped to rule- Introduction.
Invasive pulmonary aspergillosis (IPA) is a devastating disease in immunosuppressed patients. The incidence of IPA is roughly 15% among allogeneic hematopoietic stem cell transplant (HSCT) recipients and neutropenic patients with hematologic malignancies, and generally slightly lower among solid organ transplant recipients (5,7,13,15,21). Case fatality rates are as high as 50-90% despite aggressive antifungal therapy (5,11,13,21). Prompt diagnoses of IPA improve survival (6,23), but they are difficult to make due to the inadequacies of conventional diagnostic methods. At present, diagnosis generally depends upon the cultivation of Aspergillus from respiratory tract samples or the detection of hyphae within biopsy specimens. These approaches are limited by the insensitivity of cultures and the invasiveness of transbronchial biopsies. Presumptive diagnoses based on the evolution of lesions detected by thoracic CT scanning facilitate the institution of therapy in the absence of culture or biopsy results (2,3,12,24), but this strategy is limited by low sensitivity and a lack of specificity for IPA compared to other infectious processes (25). Moreover, classic halo and air-crescent ACCEPTED
signs are well-described among neutropenic hosts, but are less common in solid organ Not surprisingly, there is much interest in alternative diagnostic methods that might complement conventional approaches (9). Best studied among these is a commercially available double-sandwich ELISA that detects galactomannan (GM), a cell wall polysaccharide of most Aspergillus and Penicillium species that is released into serum during growth in tissue (Platelia ELISA, BioRad). The overall sensitivity of the serum ELISA is approximately 61%-71%, with specificity of 89%-93% (16). The test performs best among HSCT recipients and patients with hematologic malignancies, populations with the highest incidence of IPA (16). Experience among patients undergoing solid organ transplantation is much more limited. In studies of lung and liver transplant recipients, the sensitivities of the assay were 30% and 56%, respectively (4,8), with specificities of 93%-95% and 87%-94%, respectively (4,8,10). It has been suggested that the moderate sensitivity and relatively low positive predictive value of serum GM in diagnosing IPA might be improved by testing bronchoalveolar lavage (BAL) samples (8,14). Among HSCT recipients and patients with hematologic malignancies, detection of GM within BAL samples added to the sensitivity of both BAL culture and serum GM detection (1,14,17-20). While the specificity of BAL GM detection has generally been good (14,17), high rates of false positive results were reported in at least one study (22). To date, there have been no studies of BAL GM detection among solid organ transplant recipients. The objectives of ACCEPTED
this study are to review our experience with BAL GM detection among solid organ transplant recipients and to assess the utility of the assay in the diagnosis of IPA. Methods.
Identification of patients. We reviewed all cases of solid organ transplant recipients from the Shands Teaching Hospital at the University of Florida who had BAL fluid tested for GM between September 2004 and September 2006. BAL was performed according to the methods of individual pulmonologists. In general, the bronchus of the lobe in which consolidation was imaged by chest radiograph or chest CT scan was wedged, and 50 mL of 0.9% sterile saline solution at room temperature was instilled with a syringe through the working channel of the bronchoscope. The total volume of saline solution instilled into the lung was typically 150 mL, and 50 to 100 mL of BAL fluid was recovered. The BAL was sent unprocessed on dry ice via overnight mail to MiraVista Diagnostics Platelia™ Aspergillus EIA. The Platelia™ Aspergillus EIA (Bio-Rad Laboratories, Redmond, WA) was performed at MiraVista Diagnostics (Indianapolis, IN) according to the manufacturers’ procedures. Although the Platelia™ Aspergillus EIA is not FDA- cleared for testing BAL, accuracy in BAL was validated at MiraVista Diagnostics. First, 100 µL of the Platelia® treatment solution was added to 300 µL of the BAL or serum specimen, which was then heated for four minutes in a heat block (Fisher Scientific, Chicago, IL) at 104ºC, followed by centrifugation at 10,000 x g for 10 minutes. Next, 50 µL of the supernatant and 50 µL of the horseradish peroxidase-labeled monoclonal ACCEPTED
antibody (EBA-2) were incubated in the antibody pre-coated microplates for 90 minutes at 37ºC. The plates were washed five times, after which they were incubated with 200 µL of substrate chromogen reaction solution for 30 ± 5 minutes in the dark at room temperature. The reaction was stopped with sulfuric acid. Finally, within 30 minutes of adding the sulfuric acid, the plates were read at 450 nm with a reference filter of 620/630 nm. An OD index of 0.5 was considered positive. All positive samples were retested and considered positive only if the repeat test was also positive. Tests were performed as samples were received and results were reported the same day for negative specimens and after the confirmation the next day for positive specimens. Case definitions. Proven, probable and possible IPA was defined using modified EORTC-MSG criteria [http://www.doctorfungus.org/lecture/eortc_msg_rev06.htm], and assigned by physician investigators in a blinded fashion. In the event of disagreement, a consensus was reached by the investigators. BAL GM results were not made available to the investigators until the reviews were finished. The results are not included in the Definition of positive BAL GM. BAL GM results were reported as numerical values to the physicians caring for patients. The physicians made all management decisions. Interpretive cut-off values for positive BAL GM have not been established, but in this review we adopted the 0.5 cut-off proposed for serum testing. ACCEPTED
Data analysis. The sensitivity, specificity, positive and negative predictive values were calculated for BAL GM, serum GM, BAL cytology and culture. The optimal cutoff for BAL GM was determined by receiver operating characteristic (ROC) analysis. Factors associated with IPA were determined using Fisher’s exact test and expressed in 2-by-2 contingency tables; p-values <0.05 were considered significant. Results.
Description of patient population. Eighty-one solid organ transplant recipients from
our medical center had Platelia ELISA performed by MiraVista Diagnostics on BAL fluid over a two year period [Table 1]. BAL was performed for the following reasons: respiratory symptoms (n=61), fever/sepsis and abnormal imaging study of the chest (n=17), abnormal chest X-ray findings during routine clinic visit (n=2), and routine BAL surveillance following lung transplant (n=1). Five patients had IPA (liver transplant, n=1; heart transplant, n=1; kidney transplant, n=3) [Table 2]. According to modified EORTC/MSG criteria, two patients were classified as proven IPA and three as probable IPA. No patients fulfilled criteria for Performance of diagnostic tests and radiological studies.
BAL GM. Seventeen patients had at least one BAL GM 0.5 (12 patients had BAL GM ACCEPTED
1.0) [Tables 2 and 3, Figure 1]. Only one patient was receiving an agent with anti- mould activity for prophylaxis at the time of BAL collection (ABLC; 5 mg/kg); this patient had proven IPA and a GM level of 8.83 (patient #1 in Table 2). The sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of BAL GM at various interpretive cut-offs are presented in Table 4. All five patients with IPA had BAL GM levels 2.1 (range 2.1-10.12). A cut-off 0.5 yielded sensitivity and NPV of 100%, with relatively low specificity and PPV [Table 4]. Increasing the cut-off to 1 improved the specificity [Table 4]. As shown in the receiver operating curve [Figure 2], further increasing the cut-off to 1.5 and 2 improved BAL cytology and culture, and serum GM. BAL fluid was sent for cytology in 78 patients (including 4 of 5 patients with IPA) and culture in all patients. The sensitivity of cytology and culture were 50% and 40%, respectively [Table 4]. Serum GM was ordered for 38 patients, including 4 of the 5 patients with IPA. In each case, serum and BAL GMs were collected within three days of one another. Only one patient with IPA demonstrated a serum GM level 0.5 (0.93; sensitivity of serum GM: 25%). Moreover, two patients with IPA had 4 serum GMs that were negative within a week of a positive BAL GM (patients #1 and 2, Table 2). The specificity, PPV and NPV of serum GM (positive test defined as a single value 0.5) are compared to BAL GM, cytology and culture in Table 4. The concordance between serum and BAL ACCEPTED
Test results and radiological findings significantly associated with IPA. The following test results and radiological findings were more closely associated with IPA than alternative diagnoses [Table 6]: 1) BAL GM > 1.0; 2) cavitary lung lesions on chest CT scan; 3) BAL cytology consistent with mould; and 4) positive BAL culture for Aspergillus or cytology for mould. Chest X-ray and/or CT were performed in 78 patients at our medical center. We did not find any association between IPA and serum GM levels or nodules/nodular infiltrates without cavities. Indeed, nodular lesions were described in a range of diagnoses, including bacterial pneumonia (n=6), pulmonary histoplasmosis (n=2), pulmonary nocardiosis (n=2), disseminated methicillin-resistant Staphylococcus aureus (MRSA) or enterococcal infection (n=2), Rhodococcus pneumonia (n=1), cytomegalovirus pneumonitis (n=1), lung cancer (n=1) and bronchiolitis obliterans organizing pneumonia (n=1). Two patients with IPA had nodular lesions and cavities. An air-crescent sign was detected in only one patient, who was found to have pulmonary nocardiosis but not IPA. Not surprisingly, positive BAL cultures for either Aspergillus sp. or Penicillium sp. were significantly associated with BAL GM 1.0 (p=0.003). The presence of hyphal elements on cytology was also associated with BAL GM 1.0 (p=0.001). These associations were noted whether the elevated GM represented a true- or false-positive ACCEPTED
Impact of BAL GM on the time to diagnosis of IPA. In two patients with proven or
probable IPA (patients #1 and 2 in Table 2), BAL GM was the first positive test for the disease, occurring one and four weeks before a positive brain biopsy and pleural fluid culture, respectively. In a third patient (#5 in Table 2), the BAL culture revealed both A. fumigatus and Penicillium. Since cultures took several days to grow, however, a positive BAL GM shortened the time to diagnosis and the institution of antifungal therapy. In the remaining two patients with IPA, BAL GM was positive and cytology revealed hyphae Discussion.
The most notable finding of our study was that BAL GM added to the sensitivity of conventional methods for the diagnosis of IPA, while maintaining excellent specificity (90.8% at a cut-off 1.0). The sensitivity of BAL GM was 100%, compared to 50%, 40% and 25% for cytology, cultures and transbronchial biopsy results, respectively, and 25% for serum GM 0.5. In three patients (#1, 2 and 5 in Table 2), a positive BAL GM suggested IPA several days to four weeks before a diagnosis was available by other methods. Conversely, we found that a negative BAL GM effectively excluded the diagnosis of IPA (NPV=100% at a cut-off 1.0). Moreover, in the two cases of IPA in which serial bronchoscopies were performed, clinical responses to antifungal therapy were associated with decreases in BAL GM levels to < 0.5. In our experience, therefore, BAL GM was a useful adjunct to conventional tests in diagnosing, excluding and following IPA among solid organ transplant recipients. The major shortcoming of the test was false positive results, as was also reported ACCEPTED
in at least one previous study of patients with hematologic malignancies (22). In our series, the PPV was 41.7% using a cut-off 1.0 and 29.4% using a cut-off 0.5. None of the false positive tests occurred among patients receiving piperacillin-tazobactam or other antimicrobials previously linked to false positive serum results. Rather, both true- and false positive BAL GMs were significantly associated with cultures that yielded Aspergillus or Penicillium sp., and/or cytology that revealed hyphal elements. Moreover, the extent to which a GM level was positive did not differ for patients with and without IPA. These observations imply that BAL GM reflected the presence of moulds but did not distinguish between invasive disease and colonization. The performance of BAL GM among our lung transplant recipients merits particular consideration for two reasons. First, there were no cases of proven or probable IPA, fungal tracheobronchitis or bronchial anastomotic infections among patients receiving lung transplants, which precluded any assessment of the diagnostic utility of the test in this population. Second, lung transplant recipients accounted for almost half of the false positive test results (41.7% (5/12) at a cut-off 0.5, and 42.9% (3/7) at a cut-off 1.0). The high rate of false positives is not surprising. While aspergillosis has been reported in about 6% of patients receiving lung transplants, Aspergillus species can be detected in cultures of airway samples from 25%-30% of patients (21). Indeed, BAL cultures were positive for Aspergillus in three of the sixteen lung transplant recipients in this study, all of whom had extremely high GM levels (8.1, 7.14 and 3.04). If we exclude the lung transplant recipients from our analysis, the specificity and PPV among ACCEPTED
patients receiving other solid organ transplants increase to 92.9% and 62.5%, respectively Based on our data, we cannot conclusively define interpretive criteria for BAL GM. In part, this is due to the relatively small sample size and the low number of IPA cases in our study. In addition, the distribution of data limited our ability to draw conclusions about cut-offs in the 1.0-2.0 range; all five proven or probable cases were associated with levels 2.1, but only two false-positive cases exhibited levels between 1.0 and 2.0. As the cut-off was increased from 1.0 to 2.0, therefore, sensitivity did not differ and specificity improved minimally [Figure 2]. Increasing the cut-off from 0.5 to 1.0, on the other hand, was associated with more dramatic improvements in test performance, and the elimination of five false-positive cases. Since bronchoscopy is commonly utilized in the evaluation of solid organ transplant recipients with respiratory symptoms and/or abnormal findings on imaging studies, BAL GM is easy to incorporate into standard clinical practices. In addition to making more rapid diagnoses, facilitating the prompt institution of antifungal therapy and helping to rule out IPA, BAL GM might also lessen the need for invasive procedures like tissue biopsy to establish definitive diagnoses. Despite the test’s appeal, potential obstacles to its successful widespread use include a lack of standardized methodologies for collecting BAL fluid, and uncertainties about the causes of false positive results and the impact of antifungal agents on the sensitivity of the test. Clearly, issues such as optimal methodologies, interpretive criteria and the most rational use of BAL GM in ACCEPTED
widespread clinical practice merit assessment in well-designed prospective studies. Acknowledgements.
This study was supported by the University of Florida Mycology Research Unit (NIH PO1 AI061537-01 to Drs. Nguyen, Clancy and Wingard). Dr. Wheat is the President and Director of MiraVista Diagnostics, which performs BAL GM testing as a commercial References.
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Table 1. Demographics of enrolled patients.

Age (median, range)
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* 4 patients received multiple organ transplant (2 heart-lung and 2 heart-kidney); they are not listed in the lung or kidney groups. ** 1 patient received lung-kidney transplant; he is not listed in the kidney group. *** 1 patient received liver-pancreas transplant. Table 2. Clinical characteristics of patients with IPA.

Patient Transplant Reasons for CXR/ CT scan
Antibiotics
Serum - Cytology
Culture of
Diagnosis Treatment Outcome
prior to or
BAL fluid
Follow-up
at the time
BAL fluid
Resection of the brain and thyroid lesions ACCEPTED
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* transplant status: transplanted organ, time from last transplant to BAL, immunosuppressive regimen Abbreviations: ND: not done; TBBx: transbronchial biopsy; f/u: follow-up Myco: Mycophenolate; Pred: prednisone; Sol: solumedrol; FK506: tacrolimus; Siro: sirolimus; ATG: anti-thymocyte globulin; Vori: voriconazole; ABLC: amphotericin B lipid complex; Levo: levofloxacin; Mox: moxicyin; Azi: azithromycin; Tim: timemtin; CFX: ceftriaxone; TMP-SMX: trimethoprim-sulfamethoxazole. 1 Follow-up BAL during antifungal therapy: 7.72 (21 days), 0.19 (5 months), 0.26 (9 months) 2 Follow-up serum GAL over the next four days: 0.07, 0.07, 0.06 3 Follow-up BAL GAL during antifungal therapy: 0.13 (2 mos), 0.23 (9 months) 4 Follow-up serum GAL over the next six days: 0.44, 0.06, 0.06, 0.12 5 Follow-up serum GAL during antifungal therapy: 0.11 (4 days), 0.06 (1 month), 0.07 (1 month), 0.07 (2 months) ACCEPTED
Table 3. Clinical characteristics of patients without IPA but with BAL GAL > 0.5.

Patient Transplant Reasons for
CXR/ CT scan
Antibiotics
Serum - Cytology
Culture of
Diagnosis Treatment Outcome
prior to or
BAL fluid
Follow-up ** or
at the time
of BAL fluid
cause of death
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* transplant status: transplanted organ, time from last transplant to BAL, immunosuppressive regimen ** time from BAL to follow-up or death Abbreviations: ND: not done; f/u: follow-up; SOB: short of breath; MSOF: multisystem organ failure; MDR: multi-drug resistant; PCP: pneumocystis pneumonia. CyA: cyclosporin A; Myco: mycophenolate; Pred: prednisone; Sol: solumedrol; FK506: tacrolimus; Sicro: sicrolimus; Aza: azathioprine; Vori: voriconazole; Flu: fluconazole; CEF: cefipime; Vanc: vancomcyin; Azi: azithromycin; Tim: timemtin; CFX: ceftriaxone; metro: metronidazole; gati: gatifloxacin; TMP-SMX: trimethoprim-sulfamethoxazole.
Table 4. Performance of diagnostic tests.
Sensitivity
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Table 5. Concordance between serum and BAL GM levels.

(number of patients) (number of patients) (number of patients) ACCEPTED
Table 6. Factors associated with IPA.

Factors IPA
Culture positive for Aspergillus sp. Cavitary lesions observed on chest CT or ACCEPTED
Figure 1. Distribution of BAL GM results.

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Figure 2. Receiver Operating Characteristic (ROC) Curve.
1-specificity
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