ASSESSMENT OF β-D-(1→3) - Acta Medica Mediterranea

Acta Medica Mediterranea, 2014, 30: 685
ASSESSMENT OF β-D-(1→3)-GLUCAN ASSAY FOR DIAGNOSIS OF INVASIVE FUNGAL INFECTIONS
ALTAY ATALAY1, HAFIZE SAV1, AYSE NEDRET KOC1, ORHAN YILDIZ2, GONCA DEMIR1, BULENT ESER3, GOKMEN ZARARSIZ4
1
Department of Clinical Microbiology, Faculty of Medicine, Erciyes University, Kayseri - 2Department of Infectious Diseases, Faculty of
Medicine, Erciyes University, Kayseri, - 3Department of Hematology, Faculty of Medicine, Erciyes University, Kayseri, - 4Department 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-DGlucan 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 2011. 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 2002. 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
686
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 2011. 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, prob-
Altay Atalay, Hafize Sav et Al
able 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 10000 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).
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
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
9.2.0.1).
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
1
0.88
0.5
1
0.89
0.609
<0.001
(n=5)
(0.48-1.00)
(0.73-0.96)
(0.19-0.82)
(0.90-1.00)
(0.76-0.96)
Proven plus
probable
(n=23)
0.87
0.88
0.8
0.92
0.87
0.731
<0.001
(0.66-0.97)
(0.73-0.96)
(0.59-0.93)
(0.79-0.98)
(0.77-0.94)
0.687
<0.001
Proven plus
probable
plus possible
0.81
0.88
0.88
0.81
0.84
(n=43)
(0.67-0.92)
(0.73-0.96)
(0.73-0.96)
(0.67-0.92)
(0.75-0.91)
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.
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
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 (0.91-1.00), 0.91(0.830.98) and 0.86 (0.77-0.94), respectively.
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.
Assessment of β-D-(1→3)-glucan assay for diagnosis of invasive fungal infections
689
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.
Figure 2: Interactive dot diagram which displays the distribution of (1→3)- False-positivity of BG (> 80 pg/ml not relatβ-D-glucan (BG) values for groups with the cut-off point 80 pg/ml.
ed to IFI) is an important issue that decreases
the specificity of the test. False-positive BG
Discussion
reactions are known to occur in patients with renal
failure who are undergoing hemodialysis with celThe diagnosis of IFI in an immunocomprolulose membranes, in patients treated with intramised host is difficult and often missed or delayed
venous immunoglobulins or albumin preparations,
and delayed diagnosis and therapy for IFI are assoand specimens or in those exposed to gauze or other
ciated with poor outcomes(12). On the other hand,
materials that contain glucans(3, 23).
many patients for whom the presence of an IFI is
In our study none of the patients with falsesuspected are treated empirically with antifungals
positive BG results underwent hemodialysis,
that may involve the unnecessary use of potentially
received immunoglobulin, albumin, or developed
toxic and expensive drugs(13). Since microbiological
Pseudomonasm aeruginosa/Streptococcus spp. bacculture methods frequently lack sensitivity in the
teremia. Leucocyte-removing filters were previousdiagnostics of IFI, non-culture-based tests are used
ly described as a source of BG(24). We found that
for an early non-invasive diagnosis of these infecthrombocyte infusion with leucocyte-removing filtions(14). In this study, we evaluated the utility of BG
ters seemed to be responsible for the false-positive
assay (Fungitell) as a diagnostic complement for
BG results in one patient. Two patients with falseIFI. Our results suggest that BG is a beneficial
positive BG results received fungus-derived antibimarker for the diagnosis of IFI.
otics, such as piperacillin-tazobactam and imipenBG is a component of the cell walls of many
em. However some studies have shown that the
fungal organisms and the serum concentration of
administration of these antibiotics is not related
BG has been added to the EORTC/MSG biological
with BG false-positivity(25,26).
criteria for IFI other than zygomycosis and cryptoIn our limited retrospective analysis, the findcoccosis(5,7). Cuétara et al.(15) reported that, for nine
ings suggest that the serum BG level is significantly
patients with proven IFI, BG positivity was detecthigher in patients with proven, proven plus probaed at a mean of 12 days before the fungal culture
ble, and proven plus probable plus possible IFI than
was grown. In one patient, both markers appeared
in patients with the same risk factors but with no
at the same time, and in two patients, BG positivity
indications for IFI. However, randomized prospecwas detected 2 and 30 days later than the fungal
tive cost-effective assessments of the BG assay are
culture, respectively. Mohr et al.(16) reported that in
needed to determine the role of this serological
patients with proven invasive candidiasis, the first
marker in the diagnostic strategy of IFI.
positive BG was obtained at an average of 6 days
before the fungal culture was grown. In our study,
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_________
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ALTAY ATALAY
Erciyes Üniversitesi Tip Fakültesi, Tibbi Mikrobiyoloji Anabilim Dali
Melikgazi, Kayseri
(Turkey)