Bronchoalveolar galactomannan in invasive pulmonary aspergillosis: a prospective study in pediatric patients

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1 Medical Mycology, 2015, 53, doi: /mmy/myv053 Advance Access Publication Date: 30 July 2015 Original Article Original Article Bronchoalveolar galactomannan in invasive pulmonary aspergillosis: a prospective study in pediatric patients Shilan Mohammadi 1, Soheila Khalilzadeh 2, Koroush Goudarzipour 3, Maryam Hassanzad 4, Alireza Mahdaviani 5, Nahid Aarabi 6, Mihan Pourabdollah 7 and Naseh Sigari 8, 1 Pediatric Respiratory Disease Research Center, National Research Institute of Tuberculosis and Lung Disease, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 2 Professor of Pediatrics pulmonologist, Pediatric Respiratory Disease Research Center, National Research Institute of Tuberculosis and Lung Disease, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 3 Assistant Professor of Pediatrics Hematology, Pediatric Congenital Hematologic Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 4 Assistant Professor of Pediatrics pulmonologist, Pediatric Respiratory Disease Research Center, National Research Institute of Tuberculosis and Lung Disease, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 5 Associate Professor of Clinical Immunology, Pediatric Respiratory Disease Research Center, National Research Institute of Tuberculosis and Lung Disease, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 6 Pediatric Congenital Hematologic Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 7 Assistant Professor of Pathology, National Research Institute of Tuberculosis and Lung Disease, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran and 8 Associate Professor of Pulmonology, Internal Medicine Department, Medical Faculty, Kurdistan University of Medical Sciences, Sanandaj, Iran *To whom correspondence should be addressed. Naseh Sigari, No. 5, Hawin alley, Shebli Bvld, Sanandaj, Iran. Tel: ;Fax: ; naseh.sigari@muk.ac.ir Received 1 January 2015; Revised 22 May 2015; Accepted 3 June 2015 Abstract Background: Elevations in the number of immunocompromised patients in the past decade has lead to progressive increase in the incidence of Invasive Pulmonary Aspergillosis (IPA) among children; however, early diagnosis remains a challenge. Detection of galactomannan (GM) in the bronchoalveolar lavage (BAL) fluid appears to possess higher sensitivity and specificity than serum in immunocompromised adult patients but, it rarely has been investigated in pediatric patients. Methods: We performed a prospective case-control study to evaluate the efficacy of BAL GM in immunocompromised pediatric patients. Cases were subjects fulfilling the host factor criteria as defined by the EORTC/MSG and met established definitions for proven or probable IPA. Control group was patients with possible IPA in whom diagnoses C The Author Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please journals.permissions@oup.com 709

2 710 Medical Mycology, 2015, Vol. 53, No. 7 other than IPA were confirmed and patients without risk factors of IPA who underwent bronchoscopy for other diagnostic purpose. Galactomannan testing was performed on BAL fluid samples using platelia Aspergillus seroassay. Results: Sixteen cases of IPA (4 proven, 12 probable) and 54 controls (6 possible IPA and 48 no IPA) were documented according to EORTC/MSG definitions. The sensitivity and positive predictive values of BAL GM using an OD index of 0.5 were 87.5% and 93.33% respectively. We found seven cases of IPA with negative serum GM while their BAL GM was positive. Conclusion: We found high diagnostic value of BAL GM in immunocompromised pediatric patients with IPA. The lower OD index is necessary in children to avoid missing the cases of IPA in children. Key words: Aspergillosis, Galactomannan, Bronchoalveolar lavage, Immunocompromised pediatric patients. Introduction Invasive pulmonary aspergillosis (IPA) represents frequent and serious complications in immunocompromised patients [1]. Children with hematological malignancies, solid organ or hematopoietic stem cell transplantation, severe congenital and acquired immunodeficiency, long term use of corticosteroids and other immunosuppressive medications are vulnerable to this pathogen [2 4]. Elevations in the number of immunocompromised patients in the past decade have led to progressive elevations in the incidence of IPA among children [5,6]. Despite advances in treatment, mortality of IPA is significant and as many as 30% of the cases remain undiagnosed before death [7]. Hence, prompt diagnosis could improve survival [1,2,8]. Early diagnosis of IPA remains a challenge. Currently the only gold standard for true diagnosis is direct observation or culture of the hyphae in the tissue samples [9]. However, this method is often precluded by the invasiveness of procedure in critically ill patients [10]. Further confounding our ability to diagnose invasive aspergillosis is the relative insensitivity of direct cytology, culture, and polymerase chain reaction (PCR) examination of pulmonary secretions [7,11 13]. Galactomannan (GM) is a water-soluble polysaccharide cell wall component of Aspergillus species that is produced during replication and is detectable by using doublesandwich enzyme immunosorbent assay in body fluids [14]. One advantage of this test is its early detection prior to presentation of severe clinical symptoms [15]. The sensitivity and specificity of serum GM in adult patients with suspected IPA were 71 to 100% and 85 to 100%, respectively [1,16]. There are some data suggesting there may be differences in accuracy of serum GM when used in children when compared to adults [17 19]. The 4 th European Conference on Infections in Leukemia (ECLI-4) recommended that the sensitivity and specificity of this assay in children is similar to that observed in adults [20]. In the review of literature, the false positive rates in adult and pediatric patients were listed as 3 to 10% and 10 to 44%, respectively [18]. In vitro studies have shown that galactomannan is released at an earlier time with higher concentration in the bronchoalveolar lavage (BAL) fluid than in serum [21]. In addition, it appears to yield higher sensitivity and specificity than serum GM in immunocompromised adult patients [22,23]. The utility of GM EIA in BAL fluid of pediatric patients has not been investigated as extensively as in adults and has not been systematically evaluated in children to our knowledge. In a recent study in pediatrics, true additional value of BAL GM compared to serum GM was challenged [24], and another study supports a linear correlation between BAL and serum GM in immunocompromised pediatric patients [25]. Both of these studies were retrospective investigations. In the present prospective study we sought to evaluate the utility of GM in the BAL fluid of pediatric patients with suspected IPA. Materials and Methods A prospective case-control study in children with immunocompromised condition who underwent diagnostic bronchoscopy was performed at the Masih Daneshwary and Mofid hospitals (two tertiary care referral university hospitals in Tehran, Iran, with admission of children who are experiencing various pulmonary, immunologic, hematologic, and infectious diseases) from January 2013 to March The primary goal of the study was to evaluate the diagnostic performance of BAL GM for the diagnosis of IPA in immunosuppressed pediatric patients. Case definitions Cases were immunocompromised pediatric patients who matched the revised definitions for proven or probable IPA based on EORTC/MSG[26] criteria, without the use of BAL

3 Mohammadi et al. 711 GM results. In brief, proven IPA requires cytopathologic or histopathologic evidence of acute branching septate hyphae in biopsy samples or aspiration of sterile regions with evidence of tissue damage and positive result of Aspergillus culture. Probable IPA group required the presence of at least one host factor (neutrophil count< 500/μl for more than 10 days, allogeneic HSCT, consuming of glucocorticoid with dose of 0.3mg/Kg/day of prednisone equivalent for more than three weeks, treatment with immunosuppressive drugs in the last three months), one clinical feature (a dense, well-circumscribed lesion with or without air crescent sign, hallo sign or cavity on pulmonary CT scan, or tracheobronchitis evidences in bronchoscopy), and evidence of mycological infection (positive fungal culture in the sputum or BAL sample, presence of fungal elements indicating a mold in BAL sample, or positive GM in serum). Possible group consists of patients who met both host factor and clinical criteria, but the mycological evidence was absent. All other patients were considered not to have IPA. At our department, serum galactomannan testing is performed twice weekly for at-risk patients. A single serum GM OD cutoff of 0.5 was considered positive. Serum GM testing was repeated in a week interval. In our study, fiberoptic bronchoscopy for the diagnosis of IPA was performed only when a pediatric patient had host factors and clinical features of suspected IPA, but the initial serum GM testing was negative, because serum GM is considered as a probable IPA diagnostic criteria and it would be less likely to perform bronchoscopy if mycological evidence of IPA could be driven from the first serum GM testing. For performance analysis, proven and probable IPA were grouped together as IPA. Controls were two groups: (1) Immunocompromised patients who met EORTC/MSG criteria for possible IPA but had an alternative confirmed diagnosis other than IPA. (2) All non-immunocompromised pediatric patients who underwent bronchoscopy for other diagnostic reasons in the period of study (Figure 1). Exclusion criteria in both groups included prolonged usage (more than 1 week) of antifungal agents and the use of antibiotics piperacillin/tazobactam, amoxicillin/clavulanate, as a cause of false positive result of GM testing, at the time of bronchoscopy. All patients or their parents gave written informed consent. The study was approved by the Masih Daneshwari Research Center Ethics Committee. Bronchoalveolar lavage Fiberoptic bronchoscopies were done under sedation. Oxygen was flushed through the suction channel while passing the bronchoscope through the upper airway. After that, bronchoscope was gently wedged into the selected bronchus. 3ml/kg of saline was divided into three aliquots with a maximum of 20 ml per aliquot instilled and immediately withdrawn. The samples were processed for cytology assessment, bacterial and fungal smear and culture, viral and pneumocystis jiroveci screening by PCR, and GM detection. Transbronchial lung biopsy was performed wherever possible. Mycology Bronchoalveolar lavage fluid was homogenized and subjected to direct microscopy using 10% KOH and lactophenol cotton blue mount [27]. Fungal culture was done using plating of lavage sample on sabouraud dextrose agar and incubation of plates for 3 6 days at 37C. Aspergillus species was determined based on its culture and morphological characteristics and susceptibility testing was performed [28]. BAL and serum GM Platelia Aspergillus GM EIA (Bio-Rad, France) was used to measure the galactomannan of the serum and lavage samples according to the manufactures procedures. Briefly, 300 μl of serum or BAL fluid was added to 100 μl of treatment solution, boiled for three minutes at 104 Cand then centrifuged for 10 minutes in g. Next, 50 μl of supernatant and 50 μl of conjugate were mixed and incubated in microtiter plates precoated with monoclonal antibody EB-A2 for 90 minutes at 37 C. The plates were washed five times, after which they were incubated with 200 μl of tetramethylbenzadine in the dark for 30 minutes. The reaction was stopped by 100 μl of sulfuric acid and absorbance at 450 and 620 nm read using a plate reader. Positive and negative controls were included in each assay. Results were recorded as an index relative to the optical density (OD) of the cut-off control. The GM of lavage and serum was considered positive when OD index was 0.5. All positive cases were repeated in the same sample before they were considered positive. Statistics Measurable variables were described with means and standard deviation. Relevant summary diagnostic parameters, namely sensitivity, and positive predictive values were calculated using SPSS 16 software (Chicago, WA, USA). All estimations were reported with 95% confidence intervals (CI). A P value of <0.05 was considered significant.

4 712 Medical Mycology, 2015, Vol. 53, No. 7 Figure 1. Case definition flowchart Results During the study period, 84 pediatric patients underwent diagnostic bronchoscopy. According to the EORTC/MSG host factors criteria, 36 patients were immunocompromised of whom 10 cases were excluded based on exclusion criteria (seven patients with more than a week of anti-fungal treatment and three patients who received antibiotics with false-positive result of GM testing). In all 74 remaining patients, including 26 IPA suspected immunocompromised and 48 immunocompetent patients, serum GM testing was performed before bronchoscopy and BAL sample was evaluated for GM, fungal cytology, and culture. Sixteen cases of IPA (4 proven, 12 probable), 10 possible IPA, and 48 no IPA were documented according to EORTC/MSG definitions; however, four patients of possible group were removed from the study because of unclear final diagnosis and in remaining six patients another pulmonary diagnosis was confirmed, two patients had mycobacterium tuberculosis, one had bronchiolitis obliterans organizing pneumonia (BOOP), one had graft versus host disease, and one had pulmonary metastases from extrapulmonary solid tumor. Finally we had 54 control subjects. Demographic and clinical characteristics of the cases are shown in Table 1. Table 2 shows the mycological finding of patients. Seven out of 16 BAL samples of proven/probable IPA were culture-positive for Aspergillus species, and only four were positive for hyphae by microscopic analysis. In no IPA group, one subject was culture-positive, and three had positive microscopic analysis, but they did not meet host criteria and alternate pulmonary diagnoses were confirmed. Thus, the sensitivity and specificity of BAL culture were 43.7%

5 Mohammadi et al. 713 Table 1. Characteristics of 16 patients with proven and probable IPA. Antifungal therapy Age IPA Mycological evidence Hospitalization at the time of No. (yrs) Sex category for diagnosis Diagnosis Neutropenia days procedure (Days) Outcome 1. 2 M Proven Biopsy Solid tumor No 30 No Survived F Proven Biopsy Solid tumor No 20 No Survived M Proven Biopsy ALL Yes 59 Yes (3) Survived F Proven Biopsy corticosteroid therapy No 15 No Survived 5. 4 M Probable BAL culture CID Yes 14 Yes (2) Survived M Probable Second serum GM ALL Yes 16 Yes (4) Survived 7. 2 M Probable Second serum GM ALL Yes 30 Yes (5) Survived M Probable Second serum GM ALL Yes 21 Yes (3) Survived F Probable Second serum GM AML Yes 30 Yes (4) Survived F Probable %BAL cytology &culture AML Yes 21 Yes (6) Died M Probable %BAL cytology &culture ALL Yes 55 Yes (5) Survived M Probable %BAL cytology &culture Lung transplantation No 29 Yes (2) Survived M Probable Second serum GM CID No 17 No Survived F Probable Second serum GM CID Yes 11 Yes (2) Survived M Probable Second serum GM CID No 17 No Survived F Probable BAL culture Lymphoma Yes 21 Yes (3) Died Note: M: Male, F: Female, IPA: invasive pulmonary aspergillosis, AML: acute myeloid leukemia, ALL: acute lymphocytic leukemia, CID: Congenital Immunodeficiency. Table 2. Mycological findings of patients. % of patients (no./total) With IPA Without IPA Factors Proven Probable Possible No IPA %BAL cytology positive for hyphal elements 25 (1/4) 25(3/12) 0 (0/6) 7.8 (3/48) Histopathologic finding of branching septate hyphae 100 (4/4) BAL culture positive for Aspergillus sp. 50 (2/4) 41.5(5/12) 0 (0/6) 2.63 (1/48) Serum GM: OD index (2/4) 58.3 (7/12) 0 (0/6) 5.2 (2/48) BAL GM: OD index (4/4) 83 (10/12) 16.6 (1/6) 0 (0/48) OD index (4/4) 50 (6/12) 0 (0/6) 0 (0/48) and 98.15%, respectively, and for cytology were 25% and 94.44%, respectively (Table 2). Nine cases had serum GM OD index of 0.5 after initial negative test. Using BAL OD index value of 0.5, 15 patients with positive and 55 patients with negative test were found. BAL GM positive subjects in proven, probable, possible and no IPA groups were 4/4(100%), 10/12(83%), 1/6 (16.6%), and 0/48(0.00%), respectively. The only false positive result in possible group was related to a patient with liver transplantation who had the final diagnosis of BOOP. Considering only the immunocompromised patients and by excluding immunocompetent patients as controls, the sensitivity and positive predictive value of BAL GM OD index of 0.5 will be 87.5% and 93.33% respectively. Figure 2 shows the distribution of BAL OD indices in different groups of patients. Discussion In this study, we report the diagnostic performance of galactomannan in the BAL fluid of a group of immunocompromised children. To the best of our knowledge, this is one of the only studies of IPA in pediatrics being prospectively analyzed by platelia Aspergillus GM EIA in the BAL. The most notable finding of our study was that BAL GM testing yielded high sensitivity in IPA patients whose first serum GM test were negative. The results also support the lower

6 714 Medical Mycology, 2015, Vol. 53, No. 7 Figure 2. Distribution of BAL galactomannan in different groups cut-off value of BAL GM in children than in adults. Using an OD index of 0.5, a positive BAL GM strongly suggests the presence of IPA. Invasive pulmonary Aspergillosis is the most common opportunistic invasive mycosis with significant morbidity and mortality in immunocompromised patients [6]. Diagnosis of IPA remains challenging, largely because of atypical clinical presentation and relative difficulty in obtaining tissue samples. The lower sensitivity for the detection of Aspergillus hyphae in culture and cytologic examination of BAL determined in this study (43.75% and 25%, respectively) is in agreement with other published studies [11 13]. Delay is tragic for outcome and most centers develop empirical strategies but at toxic expense (especially in children) that are costly. With the understanding that earlier diagnosis of invasive aspergillosis may facilitate higher cure rates, newer diagnostic platforms that are not based on culture have been proposed and investigated. One such interesting approach is the GM EIA in the serum and BAL samples. In retrospective published studies on adults, the sensitivity of BAL GM was more than serum testing [2,12,27], but little is known about the diagnostic value of BAL GM in children. de Mole et al. [24] and Desai et al. [25] reported a sensitivity of 82.4% and 78% and specificity of 87.5% and 100% in pediatric patients, respectively. In our study, concurrent comparison of these two EIA tests was not done, because serum GM is considered as a probable IPA diagnostic criteria according to EORTC/MSG. However, an important point to mention in our study is the finding of nine patients in whom BAL GM was positive earlier than serum GM. This reinforces the importance of bronchoscopy results as a clear indicator of IPA by minimizing the risk of false negatives and reducing the morbidity and mortality by quick diagnosis. In a prospective study of IPA in adults by Hsu et al. [22], 10 patients were presented with BAL GM OD index of 0.5 while at the same time, the initial serum testing was negative and in four of them the serum GM became positive later. These findings carry important clinical implications. Due to the low side effects of bronchoscopy and the risk of delayed diagnosis of IPA, earlier performance of fiberoptic bronchoscopy in suspected pediatric patients should be considered as in some centers it is applied in adults [29]. Although the FDA has approved a cut-off index as low as 0.5 for the serum GM, there has been much controversy regarding the optimal cut-off value of the GM EIA applied to BAL. Several studies in adults reported low specificity and PPV of BAL GM using a cut-off value of 0.5 due to high frequency of false positive results, so the value recommended to define positivity has historically been 1 [2,12,22,30]. Given the increased mortality risk associated with missing the IPA cases in children, recent studies have suggested that using a lower index cut-off may detect disease earlier in its courses [24,25]. As the present study aimed to evaluate the test use in pediatric patients, an OD index of 0.5 in the BAL GM seems to yield the optimal trade

7 Mohammadi et al. 715 off, with the sensitivity and PPV of 87.5% and 93.33%, respectively, consistent with previous studies in children. Higher values, as used in adults, reduced the sensitivity and NPV significantly. The number of false positive cases in our study was much lower than other published studies [3,12]. Considering the previous studies, most of which were retrospective, elimination of factors causing false positive was not possible. In our prospective study population, patients with explicit false positive result caused by beta lactam antibiotics were excluded from the study. Over a decade of experience regarding GM has lead to sufficient knowledge of false positive cases by physicians and this should be considered in prospective studies. Prophylactic antifungal therapy may lower the fungal burden and hamper the efficacy of a test to diagnose Aspergillus species. However, the published data are controversial. While in some studies the effect of antifungal therapy on BAL GM sensitivity were not remarkable [31,32], other studies described a rapid decrease in BAL GM after initiation of therapy[33,34] and still others have shown an improvement in the sensitivity of assay [35]. We found no effect of short time antifungal therapy on assay sensitivity for BAL GM. All patients who received therapy in our study were neutropenic patients. Although some studies have reported no difference in the sensitivity of BAL GM between patients with or without neutropenia [31], in most of the other published studies the sensitivity of assay was significantly better in neutropenic patients [11,36]. Animal studies have shown higher fungal burden and angioinvasion in neutropenic samples resulted in higher GM concentration in the lung and serum [3]. In clinical practice, empiric antifungal treatment is often administrated to neutropenic immunosupressed patients suspected of having invasive fungal disease. The higher concentration of GM in the lung of these patients may explain the lesser effect of short term antifungal therapy on the sensitivity of BAL GM assay as has been found in our study. This study is one of the few prospective analyses of BAL GM in the pediatric patients. However, certain limitations must be considered. First, the number of proven cases was too low because the invasive procedures will expose these critically ill patients to some complications. Second, using immunocompetent patients without any risk of IPA as one of control groups we could not report specificity and NPV of BAL GM. Third, comparison between the sensitivity of BAL and serum GM was not possible in our study as simultaneous measurement of serum and BAL GM may be unethical. In conclusion, our prospective study in immunocompromised children found high sensitivity, and PPV of BAL GM testing when an OD index of 0.5 was used. 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