utility of the air crescent sign. Radiology. 1985;157:605-610. 4. von Eiff M, Zuhlsdorf M, Roos N, et al. Pulmonary fungal infections in patients with hematological ...
Biology of Blood and Marrow Transplantation 13:440-443 (2007) 䊚 2007 American Society for Blood and Marrow Transplantation 1083-8791/07/1304-0001$32.00/0 doi:10.1016/j.bbmt.2006.11.014
Galactomannan Antigen Enzyme-Linked Immunosorbent Assay for Diagnosis of Invasive Aspergillosis after Hematopoietic Stem Cell Transplantation Patrick C. Foy, Jo-Anne H. van Burik, Daniel J. Weisdorf Department of Medicine, University of Minnesota, Minneapolis, Minnesota Correspondence and reprint requests: Jo-Anne van Burik, MD, MMC 250, 420 Delaware St SE, Minneapolis, MN 55455 Received August 24, 2006; accepted November 15, 2006
ABSTRACT Invasive aspergillosis is difficult to diagnose in patients undergoing hematopoietic stem cell transplantation (HSCT). In 2003, a serum enzyme-linked immunosorbent assay (ELISA) test for the detection of galactomannan (a glycoprotein found on the Aspergillus cell wall) became available in the United States. In 2004, patients undergoing HSCT were screened biweekly with the galactomannan ELISA at our institution. We performed a retrospective chart review of 121 SCT patients who underwent galactomannan testing. Thirteen of the patients (10.7%) had at least 1 positive galactomannan ELISA, and 4 had multiple positive tests. When calculated in reference to a proved or probable diagnosis of aspergillosis, the galactomannan ELISA had a sensitivity of 0.50 and a specificity of 0.94. The positive predictive value was 0.46, and the negative predictive value was 0.94. Galactomannan ELISA had fewer false-positive and false-negative results in pediatric patients than in adult patients. In 4 of the 12 cases of invasive aspergillosis, the galactomannan ELISA was positive before other microbiologic evidence of aspergillosis was available. In these cases, a positive ELISA predated culture and cytologic evidence of invasive aspergillosis by a mean of 5 days (range, 1-8 days). Our findings indicate that a biweekly serum galactomannan ELISA is a highly specific diagnostic tool for detecting invasive aspergillosis in patients undergoing HSCT. When used regularly, the ELISA may allow for earlier diagnosis of invasive aspergillosis in some patients. The test is troubled by a low sensitivity and high frequency of false-negative tests. © 2007 American Society for Blood and Marrow Transplantation
KEY WORDS Aspergillus galactomannan antigen ● Enzyme-linked immunosorbent assay cell transplantation ● Invasive aspergillosis ● Pulmonary nodules
INTRODUCTION Invasive aspergillosis (IA) is a frequent and devastating complication in patients who have undergone hematopoietic stem cell transplantation (HSCT). The diagnosis of IA remains a clinical challenge. Blood, sputum, and bronchoalveolar lavage cultures have low specificity and sensitivity in detecting IA [1,2]. Radiographic findings on computed tomography (CT) of the chest are also frequently nonspecific [3,4]. In May 2003, the US Food and Drug Administration (FDA) approved a serum enzyme-linked immunosorbent assay (ELISA) test to detect the aspergillus cell wall glycoprotein known as galactomannan (GM). 440
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Similar antigen ELISAs for GM have been available in Europe since 1996 [5]. In 2001, the European Organization for the Research and Treatment of Cancer (EORTC)/National Institute of Allergy and Infectious Diseases (NIAID) consortium included a positive serum GM ELISA as part of its definition of clinical opportunistic fungal infection in HSCT and cancer patients [6]. Initial reports suggested specificity ranging from 81% to 94% and sensitivity ranging from 64% to 96% [7-9]. More recent observations suggest a high rate of false-positive tests associated with beta-lactam antibiotics, particularly piperacillintazobactam [10-16].
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Aspergillus GM antigen assay in HSCT
The aims of the present study were to better define the positive and negative predictive value of qualitative GM ELISA and to assess whether screening could lead to earlier diagnosis of IA in HSCT patients.
PATIENTS AND METHODS In 2004, all patients undergoing HSCT at the University of Minnesota Medical Center were screened with biweekly serum GM ELISAs while hospitalized. Additional testing of GM on HSCT patients outside the hospital was done as determined by clinical providers. Assays were performed on peripheral blood serum in accordance with the manufacturer’s specifications (Bio-Rad, Redmond, WA). The tests were performed on Tuesdays and Fridays in a clinical hospital laboratory. Control samples were included in each assay batch to validate performance of the ELISA. GM ELISA was reported as either positive or negative, with a cutoff of 0.5 ng/mL. Patients were retrospectively evaluated through June 30, 2005, to allow a minimum of 6 months to a maximum of 18 months follow-up. Prospectively collected data from the University of Minnesota Blood and Marrow Transplant Database, as well as all clinical data for diagnosing fungal infections, including microbiology cultures, cytology reports from bronchoalveolar lavage procedures, biopsy findings, and CT scans, were reviewed. The probability of having an invasive fungal infection at any time after HSCT was determined by EORTC/NIAID criteria at the end of the follow-up period. A patient was classified as proved IA if he or she had a sterilely obtained specimen that was culturepositive for aspergillus; as probable IA if he or she had microbiologic evidence of aspergillus infection from other specimens, including sputum, bronchoalveolar lavage, and skin biopsy; as possible IA if he or she had CT evidence of pulmonary infiltrates in the setting of high clinical suspicion for fungal infection (neutropenia, fever, or graft-versus-host disease [GVHD]); and as no risk of IA if he or she failed to meet other criteria. These probabilities were determined both with and without GM testing in each patient. Review of GM-positive cases allowed for determination of true and false-positive GM assays. Sensitivity, specificity, and positive and negative predictive values of the GM ELISA were calculated based on the clinical diagnosis of probable or proven IA infection. Secondary analysis of adult and pediatric populations was obtained with a cutoff of age 18 years at the time of transplantation. In addition, the lead time from positive GM assay to proven or probable invasive fungal infection was calculated.
A total of 71 adult and 50 pediatric patients had at least 2 GM ELISAs tested while undergoing HSCT in 2004. These patients ranged in age from 0.4 to 68.1 years (mean, 29.5 years) at the time of transplantation. Median age for the tested patients was 25.3 years; 69 of the recipients were male (57%). The source of hematopoietic stem cell grafts varied; 52 patients received 1 or more units of umbilical cord blood, 39 received sibling donor stem cells, 15 received unrelated donor stem cells, 11 received autologous stem cells, and 1 received allogeneic natural killer cells. Three patients underwent 2 consecutive (tandem) courses of myeloablative therapy (including total body irradiation) with autologous peripheral blood HSCT. RESULTS GM ELISA was tested 1523 times among the 121 patients, for an average of 12.6 tests per patient (range, 2-53 tests). After a complete retrospective review of each patient’s clinical course, 28 of the 121 patients had no clinical, radiographic, or microbiological criteria for IA. Of the remaining 93 patients, 2 had proven IA by biopsy (1 patient with pulmonary aspergillosis by open lung biopsy and 1 patient with cerebral aspergillosis by brain biopsy). Ten patients had a probable diagnosis of IA, and 81 had a possible diagnosis if IA. Thirteen of the 121 (10.7%) patients had at least 1 positive GM ELISA test. GM assays were positive in 6 of the 12 proven or probable IA patients. Seven patients who had at least 1 positive GM assay were determined not to have proven or probable IA (falsepositive GM assays): of these, 5 had possible IA, and 2 had no IA. Four of the 12 patients with proven or probable IA met the additional EORTC/NIAID criteria for invasive fungal infection by having 2 positive serum GMs. The use of screening GM changed EORTC/ NIAID classification from possible IA to probable IA in 2 patients. Clinical diagnosis of IA including and excluding GM testing is outlined in Table 1. As shown in Figure 1, the GM ELISA had a specificity of 0.94, sensitivity of 0.46, positive predictive value of 0.46, and negative predictive value of 0.94. Table 1. Clinical Certainty of IA Infection with and without the Use of the GM ELISA EORTC/NIAID Classification of Fungal Infection None Possible Probable Proven
Without Use of GM ELISA, n (%) 28 81 10 2
(23%) (67%) (8%) (2%)
With Use of GM ELISA, n (%) 28 79 12 2
(23%) (65%) (10%) (2%)
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Figure 1. Diagnostic validity of the GM ELISA.
In a subset analysis of the 50 pediatric patients, GM was truly positive in 4, falsely positive in 1, and falsely negative in 1. The pediatric group had a GM sensitivity of 0.80, specificity of 0.978, positive predictive value of 0.50, and negative predictive value of 0.978. In a subset analysis of the 71 adult patients, GM was truly positive in 2, falsely positive in 6, and falsely negative in 6. The adult group had a GM sensitivity of 0.25, specificity of 0.907, positive predictive value of 0.25, and negative predictive value of 0.907. In 4 cases, the GM ELISA was positive in patients with neutropenic fevers before any radiographic and microbiologic evidence of IA was identified. In these cases, the positive GM ELISA preceded the appearance of other evidence by 1, 4, 7, and 8 days. DISCUSSION The diagnosis of IA remains a clinical challenge in patients undergoing HSCT. Pancytopenia and coagulopathy associated with HSCT often prohibit more sensitive and specific biopsy to diagnose aspergillosis. The search for less-invasive testing for aspergillosis has been problematic. Our analysis suggests that using the serum GM ELISA for screening is a specific but not sensitive diagnostic test for detecting IA after HSCT. Our data suggest that the sensitivity of the GM ELISA is below the value of 80.7% reported before clearance by the FDA. Our sample size of 12 cases of proven/probable IA was about 1/3 that of the FDA (n ⫽ 31). Although an increased patient population would further clarify statistical limitations of the GM assay, some discrepancy between the sensitivity in our study and the FDA data can be attributed to variation in clinical practice. The differences in GM ELISA test utility in adults and children are difficult to interpret. Our results
suggest that GM ELISA may be more sensitive and specific in pediatric populations than in adults. This contradicts previous analysis of European data suggesting that GM was associated with a higher rate of false-positive results [15] and lower specificity [10] in pediatric populations. These differences may reflect the lower cutoff value of 0.5 ng/mL used in the United States, or, alternatively, they may be related to differences in clinical practice, including the use of antibiotics known to give false-positive GM ELISA findings. Part of the challenge in assessing new tests for diagnosing IA is that few patients undergo the gold standard biopsy. Our data may be limited by the fact that only 2 of 121 patients had a proved diagnosis of IA. However, our retrospective classification of proven or probable IA in 10.7% of patients is consistent with the incidence reported in other HSCT population studies [17]. EORTC/NIAID guidelines recommend initiating appropriate antifungal medications for any probable diagnosis, and by convention, most previous studies have combined probable and proven diagnosis for assessing diagnostic tests [6]. To limit the false-positive rate, 2 positive serum GM assays have been required for a probable diagnosis of invasive fungal infection according to EORTC/ NIAID criteria established in 2001 [6]. Only 3.3% of our patients had multiple positive tests. By requiring multiple positive tests supporting the clinical findings for a probable diagnosis, fewer patients are classified as being false-positive. This increases the sensitivity of the GM assay, but does so at a reduced sensitivity. Recently published data now support the use of amphotericin B or other antimold therapy for a patient with 2 consecutive positive GM ELISAs [18]. These guidelines argue that treatment for IA should be limited to those who meet highly specific diagnostic criteria for invasive fungal infection, to minimize the exposure of patients with false-positive diagnoses to toxic drug therapy. Because piperacillin-tazobactam therapy and other clinical circumstances might lead to false-positive results, these results may be reasonable, but as newer and less toxic treatments for IA have become better defined in the past 5 years, it may be more appropriate to initiate treatment with more sensitive but less specific diagnostic criteria. Requiring only 1 positive serum GM for a probable diagnosis of invasive fungal infection may be appropriate and more clinically useful and allow initiation of empirical but clinically guided use of antifungal therapy. It also may allow earlier initiation of effective treatment, which is especially important in very-high-risk populations like HSCT recipients. The most promising aspect of our study likely lies in the 4 cases in which serum GM ELISA preceded other tests in diagnosing IA. Whether biweekly or more frequent screening changes the clinical manage-
Aspergillus GM antigen assay in HSCT
ment of IA in HSCT patients is unclear. Analysis of antifungal treatments, timing of initial treatment, and mortality need to be performed before a definitive assessment of screening GM can be made. Biweekly serum GM for detecting invasive aspergillosis in SCT is a highly specific diagnostic tool. When used regularly, ELISA may allow for earlier detection of IA in some patients. The test is troubled by the low sensitivity and high frequency of falsenegative tests. Differences in the specificity and sensitivity in adult and pediatric populations warrant further investigation.
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