ASSESSMENT OF β-d-(1 3)-GLUCAN ASSAY FOR DIAGNOSIS OF INVASIVE FUNGAL INFEC- TIONS

Transcription

1 Acta Medica Mediterranea, 2014, 30: 685 ASSESSMENT OF β-d-(1 3)-GLUCAN ASSAY FOR DIAGNOSIS OF INVASIVE FUNGAL INFEC- TIONS ALTAY ATALAY 1, HAFIZE SAV 1, AYSE NEDRET KOC 1, ORHAN YILDIZ 2, GONCA DEMIR 1, BULENT ESER 3, GOKMEN ZARARSIZ 4 1 Department of Clinical Microbiology, Faculty of Medicine, Erciyes University, Kayseri - 2 Department of Infectious Diseases, Faculty of Medicine, Erciyes University, Kayseri, - 3 Department of Hematology, Faculty of Medicine, Erciyes University, Kayseri, - 4 Department of Biostatistics, Faculty of Medicine, Erciyes University, Kayseri, Turkey ABSTRACT Aim: Invasive fungal infections (IFIs) are associated with high mortality and morbidity. However, the diagnosis of IFIs in an immunocompromised host is difficult and often missed or delayed. Our aim was to investigate the feasibility of the (1 3)-β-D- Glucan assay as a diagnostic complement for IFIs. Methods: We reviewed the records of all inpatients in various units at Erciyes University s Medical Faculty Hospital (Kayseri, Turkey) who had at least one (1 3)-β-D-Glucan assay result from August 2009 to April According to the criteria of the European Organization for the Research and Treatment of Cancer/Mycoses Study Group, IFI was classified into three clinical categories: proven, probable, and possible. Serum (1 3)-β-D-Glucan was estimated using the Fungitell assay according to the manufacturer s instructions (Associates of Cape Cod, East Falmouth, Ma, USA). Optical density index 80 pg/ml was considered positive. Results: Of the 83 patients who underwent (1 3)-β-D-Glucan assay, five patients were classified as having proven IFI, 18 patients had probable IFI and 20 patients had possible IFI. The overall (proven+probable+possible) sensitivity, specificity, positive predictive value and negative predictive value of the BG assay were 81% (95% confidence interval, 67-92%), 88% (95% confidence interval, 73-96%), 88% (95% confidence interval, 73-96%), and 81% (95% confidence interval, 67-92%), respectively. Conclusions: In this study, we evaluated the utility of the (1 3)-β-D-Glucan assay as a diagnostic complement for IFIs. Our results suggest that (1 3)-β-D-Glucan is a beneficial marker for the diagnosis of IFIs. Key words: Invasive fungal infections, (1 3)-β-D-Glucan, diagnosis, non-culture methods. Received February 18, 2014; Accepted March 24, 2014 Introduction The incidence of fungal infections is increasing, especially in immunocompromised patients, and invasive fungal infections (IFIs) are associated with high mortality and morbidity (1). Delay in instituting appropriate antifungal therapy contributes to a high death rate, and conventional microbiological methods require several days and may be negative in up to 50% of patients who have systemic. Candida or Aspergillus infections (2,3). To strengthen the consistency and reproducibility of clinical and epidemiological studies on IFIs, two large study groups, the European Organization for the Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) published definitions for immunocompromised patients with cancer and hematopoietic stem cell transplants in Diagnosis of IFI is generally based on nonspecific diagnostic or radiological techniques (4,5). The (1 3)-β-D-Glucan (BG) serological diagnostic method has recently been added to the revised EORTC/MSG guideline, which was published in 2008, as a biological criterion for IFI (5-7). In both reports, IFIs are divided into three groups: possible, probable, and proven. These definitions facilitate the identification of homogeneous groups of patients in clinical studies for evaluating new drugs and management strategies (8). Rapid serological tests appear to be useful for screening high-risk hematological patients for the early diagnosis of invasive fungal infections. One of the serological tests used is BG. The concentration of BG, a major cell-wall component of various medically important fungi, with the exceptions of Cryptococcus neoformans and Zygomycetes, in the

2 686 Altay Atalay, Hafize Sav et Al serum correlates with IFIs. It has recently been added to the EORTC/MSG as a biological criterion for IFIs (5). The aim of this study was to assess the usefulness of the Fungitell BG assay (Associates of Cape Cod, Inc. (ACCI), East Falmouth, MA), which was approved by the U.S. Food and Drug Administration as an aid in the diagnosis of IFIs. Methods Patients We reviewed the records of all inpatients that were followed up in various units at the Erciyes University s Medical Faculty Hospital (Kayseri, Turkey) who had at least one BG assay result from August 2009 to August Demographic data, baseline diagnoses, and the results of relevant laboratory, microbiology, radiology, and pathology tests were obtained from patient records. Patients were classified as having proven, probable, or possible IFI, according to EORTC/MSG definition independent of BG results. Forty-three patients, with proven IFI (n=5), probable IFI (n=18) and possible IFI (n=20) were included in the study. In brief, patients were considered to have proven IFI if Aspergillus species were recovered from tissue biopsy specimen or Candida species were isolated from at least one blood culture. Patients were considered to have probable IFI if there were clinicalradiological signs and symptoms of infection and if Aspergillus species had been recovered by culture of specimens obtained from the lower respiratory tract, or galactomannan (GM) antigenemia was detected in serum (9). Possible IFI was defined as the presence of a host factor and either mycologic evidence or clinical criteria including IFI. A control group included 40 non-neutropenic patients from hematology and oncology wards without any radiological or clinical evidence of IFI. During the study period, patients who had a history of neutropenia, prolonged use of corticosteroids, who were to receive chemotherapy for hematological malignancies or who were to undergo stem-cell transplantation were screened for GM antigenemia (Platelia Aspergillus ELISA; Bio-Rad Laboratories) and BG (Fungitell kit; Associates of Cape Cod, East Falmouth, MA, USA) twice a week. Screening tests were initiated on the day of an absolute neutrophil count of fewer than 500 cells/mm3 until recovery of neutropenia. However the screening policy was limited by the stock availability of the kits (10). For patients with proven, probable or possible IFI, the highest BG level that was measured in the 10 days before the final diagnosis was included in the study. The highest BG level that was detected during hospitalization was used for patients without IFI. Information about factors related to a false-positive BG without IFI, such as a history of undergoing hemodialysis with cellulose membranes, exposure to gauze, Streptococcus spp. and Pseudomonas aeruginosa bacteremia, and of administration of albumin, intravenous immunoglobulin, blood products, or beta-lactam antibiotics, was obtained. BG assays Serum BG was estimated using the Fungitell assay according to the manufacturer s instructions (ACCI). Frozen serum specimens were thawed, vortexed, and tested in triplicate. Five-microliter aliquots were added to microplate wells and pretreated for 10 minutes at 37ºC, with 20 ml of an alkaline reagent (0.125 M KOH/0.6 M KCl). The Fungitell BG reagent was then reconstituted and dispensed according to the instructions supplied by ACCI. A microplate spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT) with Gen5 software on board was used to accomplish kinetic analysis of the microtiter plate (10). Interpretation of BG values according to the manufacturer s recommendations was as follows: < 60 pg/ml, negative; 60 to 79 pg/ml, indeterminate; and 80 pg/ml, positive. GM assays The Aspergillus GM antigen was detected by onestage immunoenzymatic sandwich microplate assay (Bio-Rad Laboratories Platelia Aspergillus EIA, Paris, France). Samples were processed as per manufacturer s instructions. Briefly, 300 μl of the test serum was added into individual 1.5 ml polypropylene tubes, and 100 μl sample treatment solution was added to each tube. After vigorous homogenization, the tubes were heated at 120ºC in a heat block for 6 minutes, followed by centrifugation of the tube at g for 10 minutes. Fifty microliters of the supernatant and 50 μl of the horseradish peroxidise-labeled monoclonal antibody (EBA-2) were incubated in the EBA-2 coated microplates for 90 ± 5 minutes at 37ºC. The plates were then washed 5 times with a microplate washer and incubated with 200 μl of substrate chromogen reaction solution for 30 ± 5 minutes in the dark at room temperature. The reaction was stopped with 100 μl sulfuric acid solution (11).

3 Assessment of β-d-(1 3)-glucan assay for diagnosis of invasive fungal infections 687 Table 1: The clinical and laboratory features of patients with invasive fungal infections. Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; ASCT, allogeneic stem-cell transplantation; BAL, bronchoalveolar lavage; BG, (1 3)-β-D-glucan; CKF, chronic kidney failure; CT, chemotherapy; F, female; GM, Galactomannan; HL, Hodgkin s lymphoma; HRCT, High Resolution Computed Tomography; ICU, intensive care unit; KLL, chronic lymphocytic leukemia; LA, lung adenocarcinoma; M, male; MM, multiple myeloma; NA, not applicable.; NHL, non-hodgkin s lymphoma; PHOD, division of pediatric hematology/oncology; WG, Wegener s granulomatosis. Positive, negative, and cut-off controls were incorporated in each assay. Results were recorded as an index relative to the mean optical density (OD) of the threshold controls (GM index = optical density sample/mean optical density of the threshold control samples). An OD index of 0.5 was considered positive. Statistical analysis To identify the IFI, for proven, proven plus probable, and proven plus probable plus possible groups, receiver operating characteristic (ROC) curves were created, and the area under the ROC curve values with 95% confidence intervals were calculated for BG values. For each group, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate diagnostic measures were calculated with 95% confidence intervals for the 80 pg/dl BG cut-off value. The kappa test was used to verify the diagnostic reliability and

4 688 Altay Atalay, Hafize Sav et Al the interactive dot plot was constructed to show the distribution of BG values for these groups in order to identify IFIs. The p<0.05 probability level was considered statistically significant. Analyses were performed using MedCalc software (Version ). Results Forty-three of the 83 patients, representing cases classified as proven, probable, or possible, and proven plus probable plus possible according to EORTC/MSG data are presented in Table 1. Of the 43 IFI cases, 5 (11.6%) were proven, 18 (41.9%) were probable, and 20 (46.5%) were possible. Of the five patients with proven IFI, three had a positive tissue culture for Aspergillus fumigatus, and two had a positive blood culture for Candida albicans and Candida pelliculosa. The overall sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate of the BG assay are presented in Table 2. Diagnostic measures Kappa test SEN (95% CI) SPE (95% CI) PPV (95% CI) NPV (95% CI) AR (95% CI) κ p Proven <0.001 (n=5) ( ) ( ) ( ) ( ) ( ) Proven plus probable (n=23) <0.001 ( ) ( ) ( ) ( ) ( ) Proven plus probable plus possible <0.001 (n=43) ( ) ( ) ( ) ( ) ( ) Table 2: Diagnostic measures and kappa test results of (1 3)- β-d-glucan to identify proven, proven plus probable and proven plus probable plus possible invasive fungal infections. AR, Accuracy rate; 95% CI, 95% confidence interval; NPV, negative predictive value; PPV, positive predictive value; SEN, sensitivity; SPE, specificity. Figure 1: Receiver operating characteristic (ROC) curves for the diagnostic performance of (1 3)-β-D-glucan (BG) for identifying proven (a), proven plus probable (b), and proven plus probable plus possible IC (c). Area under the ROC curves were 0.97 ( ), 0.91( ) and 0.86 ( ), respectively. The ROC curves for the diagnostic performance of BG for identifying proven, proven plus probable, and proven plus probable plus possible IFI and an interactive dot diagram that displays the distribution of BG values for these groups and the control group with a cut-off point of 80 pg/ml are presented in Figures 1 and 2, respectively. In patients with proven IFI, the first positive BG was detected at an average of 4 days prior to the clinical diagnosis being made, based on the date on which the BG indicated a positive result and the first culture results grew yeast or mold. There were six patients without IFI who had at least-one serum sample with a BG value > 80 pg/ml. None of the study patients were diagnosed or treated for Pneumocystis jiroveci pneumonia. Twenty-five of 43 (58.1%) patients received antifungal prophylaxis.

5 Assessment of β-d-(1 3)-glucan assay for diagnosis of invasive fungal infections 689 Figure 2: Interactive dot diagram which displays the distribution of (1 3)- β-d-glucan (BG) values for groups with the cut-off point 80 pg/ml. Discussion The diagnosis of IFI in an immunocompromised host is difficult and often missed or delayed and delayed diagnosis and therapy for IFI are associated with poor outcomes (12). On the other hand, many patients for whom the presence of an IFI is suspected are treated empirically with antifungals that may involve the unnecessary use of potentially toxic and expensive drugs (13). Since microbiological culture methods frequently lack sensitivity in the diagnostics of IFI, non-culture-based tests are used for an early non-invasive diagnosis of these infections (14). In this study, we evaluated the utility of BG assay (Fungitell) as a diagnostic complement for IFI. Our results suggest that BG is a beneficial marker for the diagnosis of IFI. BG is a component of the cell walls of many fungal organisms and the serum concentration of BG has been added to the EORTC/MSG biological criteria for IFI other than zygomycosis and cryptococcosis (5,7). Cuétara et al. (15) reported that, for nine patients with proven IFI, BG positivity was detected at a mean of 12 days before the fungal culture was grown. In one patient, both markers appeared at the same time, and in two patients, BG positivity was detected 2 and 30 days later than the fungal culture, respectively. Mohr et al. (16) reported that in patients with proven invasive candidiasis, the first positive BG was obtained at an average of 6 days before the fungal culture was grown. In our study, in patients with proven IFI, BG positivity was first detected at an average of 4 days before the fungal culture grew. The performance of BG testing for patients who have previously received a diagnosis of IFI was evaluated in some studies retrospectively, with reported sensitivity of , specificity of , PPV of , and NPV of (17 21). Lamoth et al. (22) reported that, due to low sensitivity, the BG test should be evaluated with caution and combined with clinical, radiological, and microbiological findings. In our study, the overall (proven+probable+possible) sensitivity, specificity, PPV and NPV of the BG assay were 81% (95% confidence interval, 67-92%), 88% (95% confidence intervals, 73-96%), 88% (95% confidence intervals, 73-96%), and 81% (95% confidence intervals, 67-92%), respectively. False-positivity of BG (> 80 pg/ml not related to IFI) is an important issue that decreases the specificity of the test. False-positive BG reactions are known to occur in patients with renal failure who are undergoing hemodialysis with cellulose membranes, in patients treated with intravenous immunoglobulins or albumin preparations, and specimens or in those exposed to gauze or other materials that contain glucans (3, 23). In our study none of the patients with falsepositive BG results underwent hemodialysis, received immunoglobulin, albumin, or developed Pseudomonasm aeruginosa/streptococcus spp. bacteremia. Leucocyte-removing filters were previously described as a source of BG (24). We found that thrombocyte infusion with leucocyte-removing filters seemed to be responsible for the false-positive BG results in one patient. Two patients with falsepositive BG results received fungus-derived antibiotics, such as piperacillin-tazobactam and imipenem. However some studies have shown that the administration of these antibiotics is not related with BG false-positivity (25,26). In our limited retrospective analysis, the findings suggest that the serum BG level is significantly higher in patients with proven, proven plus probable, and proven plus probable plus possible IFI than in patients with the same risk factors but with no indications for IFI. However, randomized prospective cost-effective assessments of the BG assay are needed to determine the role of this serological marker in the diagnostic strategy of IFI. References 1) Segal BH, Almyroudis NG, Battiwalla M, Herbrecht R, Perfect JR, et al. Prevention and early treatment of invasive fungal infection in patients with cancer and neutropenia and in stem cell transplant recipients in the era of newer broad-spectrum antifungal agents and diagnostic adjuncts. Clin Infect Dis 2007; 44: ) Singh N, Paterson DL. Aspergillus infections in transplant recipients. Clin Microbiol Rev 2005; 18: ) Pickering JW, Sant HW, Bowles CA, Roberts WL, Woods GL. Evaluation of a (1->3)-beta-D-glucan

6 690 Altay Atalay, Hafize Sav et Al assay for diagnosis of invasive fungal infections. J Clin Microbiol 2005; 43: ) Badiee P, Kordbacheh P, Alborzi A, Malekhoseini S, Ramzi M, et al. Study on invasive fungal infections in immunocompromised patients to present a suitable early diagnostic procedure. Int J Infect Dis 2009; 13: ) De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, et al. Revised Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Inf Dis 2008; 46: ) Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, et al. Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious Diseases. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis 2002; 34: ) Bellanger AP, Grenouillet F, Henon T, Skana F, Legrand F, et al. Retrospective assessment of β-d-(1,3)- glucan for presumptive diagnosis of fungal infections. APMIS 2011; 119: ) Cornely OA, Maertens J, Winston DJ, Perfect J, Ullmann AJ, et al. Posaconazole vs. fluonazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 2007; 356: ) Metan G, Koc AN, Atalay A, Kaynar LG, Ozturk A, et al. What should be the optimal cut-off of serum 1,3-β- D-glucan for the detection of invasive pulmonary aspergillosis in patients with haematological malignancies? Scand J Infect Dis 2012; 44: ) Ellis M, Al-Ramadi B, Finkelman M, Hendstrom U, Kristensen J, et al. Assessment of the clinical utility of serial b-d-glucan concentrations in patients with persistent neutropenic fever. J Med Microbiol 2008; 57: ) Husain S, Kwak EJ, Obman A, Wagener MM, Kusne S, et al. Prospective assessment of Platelia Aspergillus galactomannan antigen for the diagnosis of invasive aspergillosis in lung transplant recipients. Am J Transplant 2004; 4: ) Hachem RY, Kontoyiannis DP, Chemaly RF, Jiang Y, Reitzel R, Raad I. Utility of galactomannan enzyme immunoassay and (1 3)-β-D-glucan diagnosis of invasive fungal infections: low sensitivity for Aspergillus fumigatus infection in hematologic malignancy patients. J Clin Microbiol 2009; 47: ) Karageorgopoulos DE, Vouloumanou EK, Ntziora F, Michalopoulos A, Rafailidis PI, Falagas ME. β-d-glucan assay for the diagnosis of invasive fungal infections: a meta-analysis. Clin Infect Dis 2011; 52: ) Racil Z, Kocmanova I, Lengerova M, Weinbergerova B, Buresova L, et al. Difficulties in using 1,3-(beta)-D-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies-high frequency of false-positive results and their analysis. J Med Microbiol 2010; 59: ) Cuétara MS, Alhambra A, Moragues MD, Gonzalez- Elorza E, Ponton J, del Palacio A. Detection of (1 3)- beta-d-glucan as an adjunct to diagnosis in a mixed population with uncommon proven invasive fungal diseases or with an unusual clinical presentation. Clin Vaccine Immunol 2009; 16: ) Mohr JF, Sims C, Paetznick V, Rodriquez J, Finkelman MA, et al. Prospective survey of (1 3)-beta-D-glucan and its relationship to invasive candidiasis in the surgical intensive care unit setting. J Clin Microbiol 2011; 49: ) Ostrosky-Zeichner L, Alexander BD, Kett DH, Vazquez J, Pappas PG, et al. Multicenter clinical evaluation of the (1 3) beta-d-glucan assay as an aid to diagnosis of fungal infections in humans. Clin Infect Dis 2005; 41: ) Persat F, Ranque S, Derouin F, Michel-Nguyen A, Picot S, Sulahian A. Contribution of the (1 3)-{beta}-D-glucan assay for the diagnosis of invasive fungal infections. J Clin Microbiol 2008; 46: ) Koo S, Bryar JM, Page JH, Baden LR, Marty FM. Diagnostic performance of the (1 3)-beta-D-glucan assay for invasive fungal disease. Clin Infect Dis 2009; 49: ) Obayashi T, Negishi K, Suzuki T, Funata N. Reappraisal of the serum(1 3)-b-D-glucan assay for the diagnosis of invasive fungal infections-a study based on autopsy cases from 6 years. Clin Infect Dis 2008; 46: ) Senn L, Robinson JO, Schmidt S, Knaup M, Asahi N, et al. 1,3-Beta-D-glucan antigenemia for early diagnosis of invasive fungal infections in neutropenic patients with acute leukemia. Clin Infect Dis 2008; 46: ) Lamoth F, Cruciani M, Mengoli C, Lortholary O, Richardson M, et al. Third European Conference on Infections in Leukemia (ECIL-3). β-glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3). Clin Infect Dis 2012; 54: ) Ostrosky-Zeichner L. Invasive mycoses: diagnostic challenges. Am J Med 2012; 125: ) Nagasawa K, Yano T, Kitabayashi G, Morimoto H, Yamada Y, et al. Experimental proof of contamination of blood components by (1 3)-beta-D-glucan caused by filtration with cellulose filters in the manufacturing process. J Artif Organs 2003; 6: ) Marty FM, Lowry CM, Lempitski SJ, Kubiak DW, Finkelman MA, Baden LR. Reactivity of (1,3)-beta-D- Glucan assay with commonly used intravenous antimicrobials. Antimicrob Agents Chemother 2006; 50: ) Metan G, Agkus C, Buldu H, Koc AN. The interaction between piperacillin/tazobactam and assays for Aspergillus galactomannan and 1,3-beta-D-glucan in patients without risk factors for invasive fungal infections. Infection 2010; 38: Request reprints from: ALTAY ATALAY Erciyes Üniversitesi Tip Fakültesi, Tibbi Mikrobiyoloji Anabilim Dali Melikgazi, Kayseri (Turkey)