Levels of (1→3)-β-D-glucan, Candida mannan and Candida DNA in serum samples of pediatric cancer patients colonized with Candida species
BMC Infectious Diseases
Levels of (1®3)-b-D-glucan, Candida mannan and Candida DNA in serum samples of pediatric cancer patients colonized with Candida species
Eiman Mokaddas 0
Mona HA Burhamah 1
Zia U Khan 0
Suhail Ahmad 0
0 Department of Microbiology, Faculty of Medicine, Kuwait University , Safat, 13110. P. O. Box 24923 , Kuwait
1 NBK Cancer Ward, Al-Sabah Hospital , Kuwait
Background: Surveillance cultures may be helpful in identifying patients at increased risk of developing invasive candidiasis. However, only scant information exists on the effect of Candida colonization on serum levels of diagnostic biomarkers. This prospective surveillance study determined the extent of Candida colonization among pediatric cancer patients and its possible impact on serum levels of (1-3)-b-D-glucan (BDG), Candida mannan and Candida DNA. Methods: A total of 1075 swabs originating from oropharynx (n = 294), nostrils (n = 600), rectum (n = 28), groin (n = 50), ear (n = 54), and axilla (n = 49) of 63 pediatric cancer patients were cultured for the isolation of Candida spp. Patients yielding Candida spp. from any sites were considered as colonized. Serum samples were collected from patients at the time of first surveillance culture for detection of BDG by Fungitell kit and Candida mannan by Platelia Candida Ag. Candida DNA was detected by using panfungal primers and identification was carried out by using species-specific primers and DNA sequencing. Results: Seventy-five (7.6%) swab cultures from 35 (55.5%) patients yielded Candida spp. These isolates included C. albicans (n = 62), C. dubliniensis (n = 8), C. glabrata and C. tropicalis (n = 2 each) and C. krusei (n = 1). Eleven patients were colonized at three or more sites. Eight of 36 serum samples from 6 colonized patients yielded BDG values higher than the currently recommended cut-off value of ≥80 pg/ml. However, none of the serum samples yielded Candida mannan levels ≥0.5 ng/ml and PCR test for Candida DNA was also negative in all the serum samples of colonized patients. During the study period, only two colonized patients subsequently developed candidemia due to C. tropicalis. Besides positive blood cultures, C. tropicalis DNA, BDG and Candida mannan were also detected in serum samples of both the patients. Conclusions: The present study demonstrates that while mucosal colonization with Candida species in pediatric cancer patients is common, it does not give rise to diagnostically significant levels of Candida mannan or Candida DNA in serum specimens. However, BDG values may be higher than the cut-off value in some pediatric patients without clinical evidence of invasive Candida infection. The study suggests the utility of Candida mannan or Candida DNA in the diagnosis of invasive candidiasis, however, the BDG levels in pediatric cancer subjects should be interpreted with caution.
The incidence of fungal infections among cancer
patients has shown a steady increase in recent years
[1-3]. This may partly be attributed to the use of more
aggressive chemotherapeutic regimens, resulting in more
prolonged survival of these immunosuppressed patients
while they continue to remain vulnerable to invasive
fungal infections [4,5]. Although establishing an early
diagnosis for invasive mycoses is ideal for timely
administration of specific antifungal therapy, it invariably gets
delayed due to want of culture or histopathologic
evidence . Strategies are now being evolved to identify a
subgroup of high-risk patients where prophylactic
or empirical therapeutic approach could be used for
preventing development of invasive fungal infections .
Recently, Maertens et al.  proposed a preemptive
approach based on radiologic and other surrogate
markers for the early diagnosis of invasive mycoses in
high-risk patients. Surveillance cultures for determining
the Candida colonization index in high-risk patients
may be helpful in identifying patients at increased risk
of invasion and hematogenous dissemination [8-11].
However, only scant information is available on the
effect of Candida colonization on the serum levels of
BDG, Candida mannan or Candida DNA [12-14]. In
the present communication, we report results of
Candida colonization among hospitalized pediatric
cancer patients and its possible impact on serum levels of
BDG, Candida mannan, and Candida DNA.
The study was carried out in a tertiary care Pediatric
Cancer Ward, Al-Sabah Hospital, Kuwait between July
1, 2007 to December 31, 2008. Sixty-three cancer
patients, 57 (90%) with acute lymphoblastic leukemia
(ALL) and 6 (10%) with acute myeloid leukemia (AML)
were followed-up by weekly surveillance cultures for
varying periods for assessing the extent of Candida
colonization. Forty-five patients were males. Their age
ranged from 1 to 16 years. A child was considered as
colonized if Candida sp. was isolated from one or more
anatomic sites. A patient yielding Candida sp. on repeat
cultures at least from one site was considered as
persistently colonized . The study was approved by the
Ethics Committees of the Faculty of Medicine, Kuwait
University and Ministry of Health, Kuwait. Informed
consent of the patients was obtained before collecting
the clinical samples.
Isolation and identification
A total of 1075 swabs originating from oropharynx
(n = 294), nasal (n = 600), rectum (n = 28), groin (n =
50), ear (n = 54), and axilla (n = 49) of 63 pediatric
cancer patients were cultured on Sabouraud dextrose agar
supplemented with chloramphenicol (Table 1). The
Table 1 Surveillance cultures for yeast species in
pediatric cancer patients
germ tube test was performed on all the Candida spp.
isolates for the presumptive identification of C. albicans
or C. dubliniensis. Subsequently, Candida isolates were
also identified by Vitek2 yeast identification system
(BioMerieux, France). The identification of Candida
spp. isolates was also confirmed by species-specific
amplification and/or sequencing of internally transcribed
spacer (ITS) region of rDNA.
Collection of serum samples
Five ml of blood was collected in sterile BDG-free
clotting tubes and serum was separated for the detection
of (1-3)-b-D-glucan (BDG), Candida mannan and
species-specific Candida DNA at the time of surveillance
culture. The serum was kept frozen at -20°C until used.
Thirty-six serum samples from 20 colonized patients
and 11 serum samples from nine non-colonized patients
(1-3)-b-D-glucan detection in serum
The BDG levels in serum samples were determined
using a Fungitell kit (Associates of Cape Cod Inc., East
Falmouth, MA, USA) according to the procedure
described by the manufacturer. BDG levels were assayed
against a purified Pachyman standard, which included a
five-point two-fold curve ranging from 31 pg/ml to 500
pg/ml. In brief, 5 μl of serum was dispensed per well in
duplicate and pretreated with 20 μl of 0.25 M KOH and
1.2 M KCl for 10 min at 37°C. This step inactivated pro
tease and other inhibitors present in human serum. The
Fungitell BG reagent was then reconstituted and
dispensed according to the instructions supplied by the
manufacturer of the Fungitell kit. A Microplate
Spectrphotometer (Bio-Tek Instruments, Inc., Winooski, VT,
USA) with Gen5™ software onboard was used to
accomplish kinetic analysis of the microtiter plate. The BDG
value of ≥80 pg/ml was considered as positive and a
value between 60-79 pg/ml as intermediate.
Candida mannan detection in serum
Mannan antigen was measured by Platelia Candida Ag
(BioRad, Marnes La Coquette, France). The test was
performed according to the instructions of the
manufacturer. Briefly, each test serum (300 μl) was mixed with
100 μl of treatment solution and placed in a boiling
water bath for 3 minutes. After centrifugation, the
supernatant was used for further testing. Fifty-μl of the
conjugate and an equal amount of the treated serum
supernatant was introduced into micro-titer plate wells
pre-coated with anti-mannan monoclonal antibody.
After incubation at 37°C for 90 min and 5 washing
steps, 200 μl of the substrate buffer was added to each
well, and the plates were incubated for 30 min at room
temperature. The enzymatic reaction was terminated by
adding stopping solution and the optical density was
read at 450 nm using a Microplate Spectrphotometer
(Bio-Tek Instruments, Inc.). The reactions were
performed in duplicates and each experiment included
positive and negative controls as well as a calibration
curve. The calibration curve was made with a pool of
normal human serum supplemented with known
concentrations of mannan ranging from 0.1 to 2 ng/ml. A
value of ≥0.5 ng/ml was taken as positive and ≥0.25 ng/
ml but ≤0.5 ng/ml as doubtful.
Candida DNA detection by PCR
DNA from cultured Candida spp. was isolated as
described in detail previously [16,17]. DNA from serum
was extracted using the QIAamp DNA kit (QIAGEN,
Hilden, Germany) by following the instructions supplied
by the manufacturer. DNA sequences of pan-fungal and
species-specific forward and reverse primers and DNA
amplification protocol were same as described
previously [16,18]. Cultures of C. albicans ATCC 90029,
C. parapsilosis ATCC 10233, C. tropicalis ATCC 750,
C. glabrata ATCC 90030 and C. dubliniensis CBS 7987/
CD36 were used as reference for amplification of
specific products. The amplified DNA fragments were
detected by agarose gel electrophoresis using 2% agarose
gels as described previously .
The DNA isolated from selected isolates was also
subjected to direct DNA sequencing of ITS region of rDNA
(containing the ITS-1, 5.8S rRNA and ITS-2) to confirm
species-specific identification by PCR. The ITS region
was amplified by using ITS1 and ITS4 primers and both
strands of amplified DNA were sequenced as described
previously [20,21]. The sequencing primers, in addition
to the amplification primers, included ITS1FS, ITS2,
ITS3 and ITS4RS and the sequences were assembled as
described previously . GenBank basic local
alignment search tool (BLAST) searches (http://www.ncbi.
nlm.nih.gov/BLAST/Blast.cgi?) were performed for
Mann-Whitney 2-tailed test was applied to determine
the significance of differences that existed between BDG
or Candida mannan levels between different patient
groups who were colonized with Candida spp. on single
occasion or persistently and non-colonized subjects. The
Spearman correlation test was performed to determine
the correlation between BDG and Candida mannan.
A P value of <0.05 was considered as significant.
from different anatomic sites of 63 pediatric cancer
patients, 75 (7.8%) were positive for Candida spp. These
isolates included C. albicans (n = 62), C. dubliniensis
(n = 8), C. glabrata and C. tropicalis (n = 2 each) and
C. krusei (n = 1) (Tables 1-2). Apart from phenotypic
identification, all Candida spp. isolates were also
identified by species-specific amplification of ITS region of
rDNA (data not shown). The identity of six selected
isolates was further confirmed by direct DNA sequencing
of ITS region of rDNA (EMBL Accession Nos.
FN652297, FN652298, FN652301 to FN652304). The
distribution of anatomic sites yielding Candida spp. in
culture was as follows: rectum, 36%; oropharynx, 18%;
groin, 10%; ear, 2% and nasal, 1% (Table 1). Seventeen
patients were colonized at one site and 18 at two or
more sites. None of the swabs taken from axilla were
positive for Candida spp. Of the 8 C. dubliniensis
isolates, 5 came from oropharynx, and one each from nose,
groin and rectum of six patients. Two patients yielded C.
glabrata (nose and rectum) and two others yielded
C. tropicalis (oropharynx and rectal). A single isolate of
C. krusei was recovered from the oropharynx (Table 2).
In colonized patients, the mean Candida mannan
value was 0.16 ± 0.044 ng/ml, which was not
significantly different from those that were not colonized (p =
0.660) (Table 3). There was also no significant difference
in the mean Candida mannan values between patients
colonized at one anatomic site or two or more sites (p =
0.665) or between those that were colonized once or
those yielding Candida spp. repeatedly (p = 0.474)
(Table 3). Although mean BDG values in colonized
patients were nearly same as non-colonized patients
(46.98 pg/ml vs. 36.77 pg/ml), 8 serum samples from
6 colonized patients were positive for BDG (range 82
pg/ml to 141 pg/ml, mean = 98.3 pg/ml). Three of these
serum samples were obtained from the same patient
within a span of 40 days and the BDG levels varied
between 85 pg/ml to 115 pg/ml. However, none of these
8 serum samples yielded mannan levels >0.25 ng/ml.
Additionally, 5 serum samples from two colonized
patients yielded BDG values in the intermediate range
(Table 3). There was no significant difference in the
mean BDG values between patients that were colonized
once or those that yielded Candida spp. persistently
(p = 1.0) (Table 3). The mean and standard deviation of
BDG and Candida mannan levels in serum samples
collected from the patients at the time of surveillance
samples were 42.74 ± 30.18 pg/ml and 0.173 ± 0.03 ng/ml,
respectively. No correlation was observed between BDG
and Candida mannan values among patients that were
colonized with Candida species by Spearman correlation
test (p = 0.531, R = 0.108). None of the serum sample
from the colonized patients was positive for the
detection of Candida DNA by PCR.
Table 2 Species spectrum of Candida species isolated from different anatomic sites of pediatric cancer patients
Two patients whose oropharyngeal or rectal samples
yielded C. tropicalis (in addition to C. dubliniensis in one
and C. albicans in the other) developed candidemia due
to C. tropicalis. Besides blood culture positivity, both
these patients showed presence of C. tropicalis DNA in
serum by species-specific PCR amplification (Fig. 1) and
also showed elevated levels of BDG (238.6 and 406 pg/ml),
whereas Candida mannan was positive in one (0.77
ng/ml) and border line (0.28 ng/ml) in the other.
Infections caused by Candida spp. are the major cause
of morbidity and mortality among seriously ill patients.
Prior colonization with Candida spp. has been regarded
as an essential step for the development of invasive
disease [10,23]. The colonization index could be helpful in
predicting risk of developing systemic infection in
critically ill patients, and thus, may offer opportunities for
early therapeutic or prophylactic interventions [6,10]. In
the present study, although 11 of 35 (31%) patients were
colonized with Candida spp. At ≥3 sites, none of them,
despite being leukemic, developed candidemia or
invasive candidiasis. Two patients with ALL who
subsequently developed candidemia due to C. tropicalis were
colonized at two sites with two different Candida spp.
(C. albicans with C. tropicalis or C. dubliniensis with
C. tropicalis) and their serum samples also yielded
positive results for C. tropicalis DNA, mannan and BDG.
Recently, Leon et al.  conducted a prospective
observational study in a cohort of non-neutropenic patients
to assess the value of “Candida score” for the
probability of developing invasive candidiasis. Since invasive
candidiasis occurred only in <5% of patients who had
Candida colonization score of <3, the likelihood of
developing invasive candidiasis in such patients was
considered very low.
Since early diagnosis of invasive candidiasis is
challenging, the role of surrogate markers, such as Candida
species-specific DNA, mannan, and BDG in predicting
the onset of invasive candidiasis has attracted
considerable attention [13,24]. None of the colonized patients in
the present study were found positive for Candida
mannan or Candida DNA. However, BDG levels were
positive in eight serum samples from six patients with
values ranging from 82 pg/ml to 141 pg/ml. These
observations are generally in agreement with previous
studies showing that patients colonized at single or
multiple sites yield BDG levels below the cut-off value
recommended by the manufacturer [12,25-27]. The
BDG levels above the cut-off values (80 pg/ml) in 6 of
20 (30%) colonized pediatric cancer patients in our
study may either result from absorption of BDG through
the gut due to mucositis  or due to contamination
with cellulose , gauze , bacterial sepsis [30,31] or
intravenous therapy with amoxicillin-calvulanic acid in
these subjects . Despite the above limitations of the
test, several studies have used BDG monitoring to
identify patients at risk of developing invasive candidiasis to
improve therapeutic outcome [25,33,34]. A wide range
of sensitivities and specificities have been obtained in
different study populations [25,34,35], probably due to
use of different cut-off values for a positive BDG test or
due to use of different brand of kits that may react
differently to BDG present in the clinical samples [36,37].
Unlike BDG, Candida mannan levels in serum seem to
be less susceptible to the extent of Candida
colonization. As stated above, none of our colonized patient was
found to have positive serum levels (>0.5 ng/ml) for
Table 3 (1-3)-b-D-glucan and Candida mannan levels in serum samples of different groups of patients
No. of patients No. of samples
GM, Geometric mean; SD, Standard deviation
Figure 1 Agarose gel showing amplification of a DNA
fragment of ~106 bp by seminested PCR with DNA isolated
from serum from patient 1 (lane 2) and patient 2 (lane 3). An
amplicon of the same size was also obtained with genomic DNA
isolated from reference strain of C. tropicalis (lane 4) while no
amplicon was obtained in the reagent control tube in which water
instead of DNA was added (lane 1). Lane M is 100 bp DNA marker
and the positions of migration of 100 bp, 200 bp and 600 bp
fragments are marked.
Candida mannan (mean 0.16 ± 0.04 ng/ml). In two
patients who had Candida mannan in the intermediate
range (0.308 and 0.287 ng/ml), sera were negative for
BDG as well as Candida DNA. These findings are in
conformity with the results of previous studies [12,14].
The normal BDG values in healthy pediatric
population are not yet established. In a preliminary study,
Brian Smith et al.  estimated BDG levels in serum
samples from 120 non-immunocompromised children
(0.5 to 18 year-old) and found higher levels (mean 68 ±
128 pg/ml) than those reported earlier in adult
population [25,35]. A noteworthy observation of this study was
that 18 of 120 (15%) children had BDG levels >80 pg/ml
and 8 of 120 (7%) had BDG levels between 60-79 pg/ml
. Thus, higher BDG levels in a minority (6 of 20,
30%) of pediatric cancer patients in our study is
consistent with the BDG data reported by Brian Smith et al.
. These observations warrant further studies in
pediatric population for establishing BDG cut-off values
for a positive test to validate the diagnostic utility of this
marker for invasive fungal infections.
This prospective surveillance study revealed that while
nearly half (55%) of the pediatric cancer patients were
colonized with Candida spp. at one or more anatomic
sites, the serum levels for Candida mannan remained
significantly less than the cut-off value recommended by
the manufacturer for a positive test. Also, Candida
DNA was not detected in the serum samples of the
colonized patients by the semi-nested PCR used.
However, BDG levels were elevated in a minority of
colonized pediatric patients suggesting that additional
evidence of infection may be needed for the diagnosis of
invasive candidiasis in this patient population.
BDG: (1-3)-b-D-glucan; PCR: Polymerase Chain Reaction; DNA:
Deoxyribonucleic Acid; ALL: Acute Lymphoblastic Leukemia; AML: Acute
Myeloblastic leukemia; KOH: Potassium Hydroxide; KCl: Potassium Chloride;
ITS: Internally Transcribed Spacer; CBS: Centraalbureau voor Schimmeculture.
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