diagnostyka molekularna kandydozy markery.pdf

Mokaddas et al. BMC Infectious Diseases 2010, 10:292

http://www.biomedcentral.com/1471-2334/10/292

RESEARCH ART ICLE

Open Access

-D-glucan, Candida mannan

and Candida DNA in serum samples of pediatric

cancer patients colonized with Candida species

3)-

Eiman Mokaddas 1* , Mona HA Burhamah 2 , Zia U Khan 1 , Suhail Ahmad 1

Abstract

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)-

-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

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.

≥

Background

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

whiletheycontinuetoremainvulnerabletoinvasive

fungal infections [4,5]. Although establishing an early

diagnosis for invasive mycoses is ideal for timely admin-

istration of specific antifungal therapy, it invariably gets

delayed due to want of culture or histopathologic evi-

dence [5]. Strategies are now being evolved to identify a

subgroup of high-risk patients where prophylactic

or empirical therapeutic approach could be used for

* Correspondence: e.mokaddas@hsc.edu.kw

1 Department of Microbiology, Faculty of Medicine, Kuwait University, Safat,

13110. P. O. Box 24923, Kuwait

Full list of author information is available at the end of the article

Commons Attribution License ( http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly cited.

Levels of (1

yielded Candida mannan levels

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preventing development of invasive fungal infections [6].

Recently, Maertens et al. [7]proposedapreemptive

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 can-

cer patients and its possible impact on serum levels of

BDG, Candida mannan, and Candida DNA.

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.

-D-glucan (BDG), Candida mannan and spe-

cies-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

were tested.

Methods

Study population

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 persis-

tently colonized [15]. 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.

(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

l 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 dis-

pensed according to the instructions supplied by the

manufacturer of the Fungitell kit. A Microplate Spectr-

photometer (Bio-Tek Instruments, Inc., Winooski, VT,

USA) with Gen5

software onboard was used to accom-

plish kinetic analysis of the microtiter plate. The BDG

value of

™

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 can-

cer patients were cultured on Sabouraud dextrose agar

supplemented with chloramphenicol (Table 1). The

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 manufac-

turer. Briefly, each test serum (300

Table 1 Surveillance cultures for yeast species in

pediatric cancer patients

Sample site

l) was mixed with

l of treatment solution and placed in a boiling

water bath for 3 minutes. After centrifugation, the

supernatant was used for further testing. Fifty-

No. positive/No. samples (%)

lofthe

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

Oropharyngeal

53/294 (18)

Nasal

6/600 (1)

Rectal

10/28 (35.7)

Groin

5/50 (10)

Ear

1/54 (1.9)

l 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

Axilla

0/49 (0)

Total

75/1075 (7)

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)-

l of serum was dispensed per well in

duplicate and pretreated with 20

100

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adding stopping solution and the optical density was

read at 450 nm using a Microplate Spectrphotometer

(Bio-Tek Instruments, Inc.). The reactions were per-

formed in duplicates and each experiment included

positiveandnegativecontrolsaswellasacalibration

curve. The calibration curve was made with a pool of

normal human serum supplemented with known con-

centrations 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/

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 identi-

fied by species-specific amplification of ITS region of

rDNA (data not shown). The identity of six selected iso-

lates 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%andnasal,1%(Table1).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 iso-

lates, 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 signifi-

cantly 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 col-

lected from the patients at the time of surveillance sam-

ples 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 detec-

tion of Candida DNA by PCR.

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 pre-

viously [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 speci-

fic products. The amplified DNA fragments were

detected by agarose gel electrophoresis using 2% agarose

gels as described previously [19].

The DNA isolated from selected isolates was also sub-

jected 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 [22]. GenBank basic local align-

ment search tool (BLAST) searches ( http://www.ncbi.

nlm.nih.gov/BLAST/Blast.cgi?) were performed for spe-

cies identification

Statistical analysis

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.

Results

Surveillance cultures

The results of the surveillance cultures for Candida spp.

are presented in Tables 1-2. Of 1075 swabs cultured

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Table 2 Species spectrum of Candida species isolated from different anatomic sites of pediatric cancer patients

Site

C. albicans

C. dubliniensis

C. glabrata

C. tropicalis

C. krusei

Oropharyngeal

Nasal

Rectal

Groin

Ear

Total

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.

developing invasive candidiasis in such patients was con-

sidered very low.

Since early diagnosis of invasive candidiasis is challen-

ging, the role of surrogate markers, such as Candida

species-specific DNA, mannan, and BDG in predicting

the onset of invasive candidiasis has attracted consider-

able attention [13,24]. None of the colonized patients in

the present study were found positive for Candida man-

nan or Candida DNA. However, BDG levels were posi-

tive 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 mul-

tiple 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 [24] or due to contamination

with cellulose [28], gauze [29], bacterial sepsis [30,31] or

intravenous therapy with amoxicillin-calvulanic acid in

these subjects [32]. Despite the above limitations of the

test, several studies have used BDG monitoring to iden-

tify 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 dif-

ferently 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 coloniza-

tion. As stated above, none of our colonized patient was

found to have positive serum levels (>0.5 ng/ml) for

3sites,noneofthem,

despite being leukemic, developed candidemia or inva-

sive candidiasis. Two patients with ALL who subse-

quently developed candidemia due to C. tropicalis were

colonizedattwositeswithtwodifferentCandida spp.

(C. albicans with C. tropicalis or C. dubliniensis with

C. tropicalis) and their serum samples also yielded posi-

tive results for C. tropicalis DNA, mannan and BDG.

Recently, Leon et al. [9] conducted a prospective obser-

vational study in a cohort of non-neutropenic patients

to assess the value of

≥

for the probabil-

ity of developing invasive candidiasis. Since invasive can-

didiasis occurred only in <5% of patients who had

Candida colonization score of <3, the likelihood of

“

Candida score

”

Table 3 (1-3)- b -D-glucan and Candida mannan levels in serum samples of different groups of patients

Colonization Status No. of patients No. of samples

BDG (pg/

GM ± SD

Mannan (ng/ml)

GM ± SD

≥ 80 <80 - ≥ 60 <60

≥ 0.5 <0.5 - ≥ 0.25 <0.25

Colonized

23 46.98 ± 30.22 -

34 0.166 ± 0.044

Persistently colonized

12 46.60 ± 29.30 -

19 0.169 ± 0.046

One-time colonized

11 47.45 ± 32.30 -

15 0.164 ± 0.044

Non-colonized

11 36.77 ± 9.96 -

10 0.161 ± 0.06

GM, Geometric mean; SD, Standard deviation

Discussion

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 dis-

ease [10,23]. The colonization index could be helpful in

predicting risk of developing systemic infection in criti-

cally 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

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DNA was not detected in the serum samples of the

colonized patients by the semi-nested PCR used. How-

ever, BDG levels were elevated in a minority of colo-

nized pediatric patients suggesting that additional

evidence of infection may be needed for the diagnosis of

invasive candidiasis in this patient population.

-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.

Acknowledgements

Excellent technical support from Ajmal Theyyathel and Josephin Jhonson

is thankfully acknowledged. The study was supported by KFAS grant No.

2005-130-205.

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.

Author details

1 Department of Microbiology, Faculty of Medicine, Kuwait University, Safat,

13110. P. O. Box 24923, Kuwait.

2 NBK Cancer Ward, Al-Sabah Hospital, Kuwait.

Authors ’ contributions

EM, MHB, and ZUK: Conceived and supervised the study. SA supervised the

molecular part of the study. All the authors contributed to the writing and

finalizing the manuscript.

Competing interests

The authors declare that they have no competing interests.

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 popula-

tion are not yet established. In a preliminary study,

Brian Smith et al. [38] 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 popula-

tion [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

[38]. Thus, higher BDG levels in a minority (6 of 20,

30%) of pediatric cancer patients in our study is consis-

tent with the BDG data reported by Brian Smith et al.

[38]. 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.

Received: 20 January 2010 Accepted: 6 October 2010

Published: 6 October 2010

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Conclusions

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

Abbreviations

BDG: (1-3)-

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