Enzymatic differentiation of Candida parapsilosis from other Candida spp. in a membrane filtration test
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Journal of Microbiological Methods 53 (2003) 11 – 15
www.elsevier.com/locate/jmicmeth
Enzymatic differentiation of Candida parapsilosis from
other Candida spp. in a membrane filtration test
Tiene G.M. Bauters a, Renaat Peleman b, Marc Dhont c,
Piet Vanhaesebrouck d, Hans J. Nelis a,*
a
Laboratory for Pharmaceutical Microbiology, Department of Pharmaceutical Analysis, Ghent University, Harelbekestraat 72,
B-9000 Ghent, Belgium
b
Department of Internal Medicine, Division of Infectious Diseases, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium
c
Department of Obstetrics and Gynecology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium
d
Neonatal Intensive Care Unit, Department of Paediatrics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium
Received 21 May 2002; received in revised form 24 July 2002; accepted 30 September 2002
Abstract
A previously reported enzyme assay on a membrane filter using 4-methylumbelliferyl (4-MU)-N-acetyl-h-D-galactosaminide,
-phosphate and -pyrophosphate as substrates for the differentiation of four Candida spp. has been extended to Candida
parapsilosis. The substrate 4-MU-h-D-glucoside was hydrolyzed by 28 test strains of this species but to a variable extent by
seven other yeasts also. For a full enzymatic differentiation of C. parapsilosis from other medical yeasts, a battery of six
reactions was required. Of 71 C. parapsilosis positive clinical samples, 4.2% gave a false negative result due to overgrowth by
Candida albicans. The present assay is more rapid than a described spectrofluorometric determination of h-D-glucosidase in a
broth, i.e., 9 – 11 h versus up to >48 h.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Candida parapsilosis; h-D-Glucosidase; Membrane filtration
1. Introduction limited number of key enzymatic activities for the
differentiation of Candida and Cryptococcus spp. As
In recent years, non-albicans Candida species such substrates, they used fluorogenic 4-methylumbelli-
as Candida parapsilosis are increasingly implicated in feryl (4-MU) derivatives. Based on the differential
nosocomial infections, notably in cardiac surgery pa- reactions of yeast isolates with 4-MU-phosphate, 4-
tients and neonates (Moran et al., 2002; Weems, 1992). MU-pyrophosphate and 4-MU-h-D-glucoside at acidic
Candida species contain a range of hydrolytic pH, they were able to distinguish between Cryptococ-
enzymes that are useful for their identification in cus spp., Candida tropicalis, Candida glabrata and
clinical samples. Bobey and Ederer (1981) used a Candida albicans/Candida krusei. At acidic pH, C.
parapsilosis was 4-MU-h-D-glucoside negative.
* Corresponding author. Tel.: +32-9-2648091; fax: +32-9-
However, at neutral pH, Smitka and Jackson
2648195. (1989) found C. parapsilosis to be strongly 4-MU-
E-mail address: Hans.Nelis@rug.ac.be (H.J. Nelis). h-D-glucoside positive. Some other Candida spp. also
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 7 0 1 2 ( 0 2 ) 0 0 2 1 2 - 912 T.G.M. Bauters et al. / Journal of Microbiological Methods 53 (2003) 11–15
cleaved this substrate but at a much lower rate. To C. glabrata (n = 39), C. krusei (n = 25), C. tropicalis
avoid false positives, the inoculum was standardized (n = 26), C. lusitaniae (n = 4), C. dubliniensis (n = 1),
and fluorescence was read in a spectrofluorometer as a C. kefyr (n = 2), C. guilliermondii (n = 2), Cryptococ-
function of time. A reading above a defined threshold cus neoformans (n = 17), Saccharomyces cerevisiae
value indicated C. parapsilosis with allegedly 100% (n = 4), Geotrichum candidum (n = 4) and Tricho-
specificity. However, only a limited number of other sporon cutaneum (n = 4). C. parapsilosis and C.
Candida spp. was tested for possible interference. dubliniensis strains were obtained from the Institute
Although the test itself is rapid (30 – 240 min), it of Hygiene and Epidemiology, Mycology (IHEM)
requires preliminary culturing of the yeasts for 24 – (Brussels, Belgium) (C. parapsilosis IHEM 2305,
48 h to prepare the heavy inoculum required. Hence, 4224, 2052, 1716, 4223, 4024, 4606 and 6395, and
the ‘‘real’’ analysis time is >1– 2 days. C. dubliniensis IHEM 14280). Other Candida and
To reduce the test time of the detection of enzymes non-Candida yeast isolates were from our own col-
in Candida spp., the approach of Bobey and Ederer lection, as previously described (Bauters et al., 1999).
has been previously adapted to a membrane filter
format (Bauters et al., 1999). The sample is filtered 2.2. Clinical samples
over a nylon membrane filter, which is subsequently
incubated on a modified Sabouraud dextrose agar to Clinical C. parapsilosis positive samples included
yield microcolonies. In a second step, the membrane whole blood (n = 14), rectal (n = 26), oropharyngeal
filter is cut in four quarters which are incubated for 30 (n = 15), vaginal (n = 11) and finger swabs (n = 5).
min on a glass fiber pad impregnated with enzyme They were obtained from the Departments of Internal
substrates, i.e., 4-MU-N-acetyl-h-D-galactosaminide, Medicine, Division of Infectious Diseases; Obstetrics
4-MU-phosphate and 4-MU-pyrophosphate or no and Gynecology, and from the Neonatal Intensive
substrate, and digitonin acting as a membrane perme- Care Unit from the Ghent University Hospital.
abilizer. C. albicans, C. tropicalis and C. krusei are
differentiated based on the presence/absence of blue 2.3. Filters, growth media, chemicals and reagents
fluorescence on the first three parts of the filter. C.
glabrata does not hydrolyze any of the substrates but Nylon membrane filters (47 mm, 0.45-Am pore
was found to exhibit orange fluorescence in the size) used for filtration of the samples and absorbent
absence of a substrate, on the fourth part of the filter. glass fiber pads were purchased from Gelman Scien-
As C. parapsilosis was not included in the original ces (Ann Arbor, MI, USA).
test, the purpose of the present work was to investigate Sabouraud glucose agar (SGA) (Difco Laborato-
whether the addition of 4-MU-h-D-glucoside to the ries, Detroit, MI, USA) was supplemented with 5000
battery of enzyme substrates would be capable of dif- Ag/ml of a combination of ticarcillin (4688 Ag/ml) and
ferentiating C. parapsilosis, C. albicans, C. glabrata, clavulanic acid (312 Ag/ml) (TimentinR) (SmithKline
C. krusei and C. tropicalis under the conditions of the Beecham Pharma, Genval, Belgium) (SGA-T). Other
membrane filtration procedure. In addition, in view of a growth media were CHROMagar CandidaR (CHRO-
recent report about the use of h-D-glucosidase activity Magar, CHROMagar Company, Paris, France) and
to distinguish between C. albicans and C. dubliniensis cornmeal agar (Difco Laboratories) supplemented
(Sullivan and Coleman, 1998), an isolate of C. dublin- with 0.5% Tween 80.
iensis was also subjected to the current test conditions. For the differentiation of Candida spp., five sub-
strates were used, i.e., 4-MU-h-D-glucoside, 4-MU-
phosphate, 4-MU-h-D-galactoside (all from Sigma, St.
2. Materials and methods Louis, MO, USA), 4-MU-N-acetyl-h-D-galactosami-
nide and 4-MU-pyrophosphate (both from Melford
2.1. Test organisms Laboratories, Ipswich, UK). Of each substrate, 1 mg
was dissolved in 1 ml of dimethylsulfoxide (DMSO)
Yeasts used to test sensitivity and specificity in- (Fluka, Buchs, Switzerland). This stock solution was
cluded C. parapsilosis (n = 28), C. albicans (n = 129), further diluted in 0.1 M phosphate buffer, pH 7.0 (4-T.G.M. Bauters et al. / Journal of Microbiological Methods 53 (2003) 11–15 13
MU-h-D-glucoside), in 0.1 M citrate – phosphate buf- (API 20C AUX) and agglutination with the Krusei-
fer, pH 4.5 (4-MU-N-acetyl-h-D-galactosaminide, 4- ColorR test. All cultures identified as C. albicans
MU-pyrophosphate and 4-MU-galactoside) or in 0.1 were subcultured on SGA at 45 jC to distinguish
M citrate – phosphate buffer, pH 3.4 (4-MU-phos- them from C. dubliniensis, which, unlike C. albicans,
phate) to a final concentration of 0.01%. The working is unable to grow at 45 jC (Pinjon et al., 1998).
solutions were sterilized by filtration over Nalgene
disposable units (0.45-Am pore size, 250 ml) (Nalge,
Rochester, NY, USA), dispensed in test tubes and 3. Results
frozen at 20 jC until use.
Triton X-100 and digitonin were obtained from All 28 test strains of C. parapsilosis were 4-
Merck (Darmstadt, Germany) and Sigma, respectively. MU-h-D-glucoside positive. However, as shown in
API 20C AUX strips came from bioMérieux Vitek Table 1, other Candida spp. and non-Candida
(Hazelwood, MO, USA) and a latex-agglutination test yeasts also cleaved this substrate. To ensure specif-
kit (Krusei-ColorR) from Fumouze (Levallois Perret, icity of the test for C. parapsilosis, the conversion
France). of 4-MU-h-D-glucoside had to be combined with
that of four additional substrates, i.e., 4-MU-N-
2.4. Procedure acetyl-h- D-galactosaminide, 4-MU-phosphate, 4-
MU-pyrophosphate and 4-MU-h-D-galactoside, as
All clinical specimens were processed within 30 well as with one unelucidated reaction leading to
min after arrival. Swabs were extracted by vortex orange fluorescence (Bauters et al., 1999). This
mixing in 10 ml of buffered peptone (Difco Labora- combination allowed a full differentiation of C.
tories). Blood samples were pretreated with 50 Al parapsilosis from seven other Candida spp. and
Triton X-100 per ml of blood for 30 min at 30 jC. four non-Candida yeasts (Table 2).
Half of each sample was filtered over a nylon The method has been applied to 71 clinical
membrane filter which was subsequently placed on samples containing C. parapsilosis as a single spe-
SGA-T and incubated for 9– 11 h at 37 jC to yield cies (n = 68) or in a mixed flora (n = 3). An agree-
microcolonies. After incubation, the filter was ex- ment of 95.8% with conventional identification
posed to UV light to observe a possible orange methods was obtained for the target species. Only
fluorescence, indicating the presence of C. glabrata. in the three samples (4.2%) with mixed flora C.
The filter was subsequently removed from the SGA- parapsilosis was overlooked due to its overgrowth
T medium and cut in five pieces. These were placed
on absorbent glass fiber pads, each one impregnated
with 340 Al of a buffered solution of the 4-MU
substrate (4-MU-h-D-glucoside, 4-MU-N-acetyl-h-D- Table 1
4-MU-h-D-glucoside positivity in yeast species in the enzymatic
galactosaminide, 4-MU-phosphate, 4-MU-pyrophos- membrane filtration test
phate and 4-MU-h-D-galactoside), containing 0.1%
Number of strains tested % Reactive strains
digitonin, acting as a membrane permeabilizer and 1
mM MgCl2. After incubation for 30 min at 30 jC, C. parapsilosis 28 100
C. albicans 129 9
the filters were sprayed with 1.2 M sodium hydrox- C. glabrata 39 8
ide and inspected under a 366-nm UV lamp. Blue C. krusei 25 0
fluorescent microcolonies indicated hydrolysis of a C. tropicalis 26 0
substrate. C. dubliniensis 1 0
The remaining part of the sample was filtered C. lusitianiae 4 25
C. kefyr 2 0
over a second nylon membrane filter which was C. guilliermondii 2 0
incubated on CHROMagar for 24– 48 h at 37 jC. Cr. neoformans 17 88
Confirmation of presumably identified colonies S. cerevisiae 4 50
relied on the germ tube test, morphology on cornmeal G. candidum 4 50
agar with 0.5% Tween 80, sugar assimilation tests T. cutaneum 4 5014 T.G.M. Bauters et al. / Journal of Microbiological Methods 53 (2003) 11–15
Table 2
Combined positive and negative reactionsa for the differentiation of C. parapsilosis from other medical yeasts
(A) Differentiation of C. parapsilosis from other Candida spp.
Reaction C. parapsilosis C. albicans C. glabrata C. krusei C. tropicalis C. guilliermondii C. lusitaniae C. kefyr
(n = 28) (n = 129) (n = 39) (n = 25) (n = 26) (n = 2) (n = 4) (n = 2)
h-D-Glucosidase + +/ +/ +/
c
N-acetyl-h-D- + +/
galactosaminidaseb
Acid phosphataseb +c +/ +/ + +/ + +
Pyrophosphataseb +/ c
+/ +
b
Unknown + + +
mechanismb,d
h-D-Galactosidaseb +c + + +
(B) Differentiation of C. parapsilosis from other yeasts
Reaction C. parapsilosis Cr. neoformans S. cerevisiae G. candidum T. cutaneum
(n = 28) (n = 17) (n = 4) (n = 4) (n = 4)
h-D-Glucosidase + + + + +
c c
N-acetyl-h-D- +
galactosaminidaseb
Acid phosphataseb +c +c + +
Pyrophosphataseb +/ c c
c c
Unknown
mechanismb,d
h-D-Galactosidaseb +c c
+
a
+/ indicates that only part of the strains are positive.
b
Bauters et al. (1999).
c
This study and previous study (Bauters et al., 1999).
d
Leading to orange fluorescence.
by C. albicans. An isolate of C. dubliniensis yielded parapsilosis but that the four other Candida spp. they
no fluorescence in the current test. examined exhibited a lower activity. To avoid false
positive results, they proposed a semiquantitative
approach using spectrofluorometry in connection with
4. Discussion cut-off values for interpretation.
In the membrane filtration approach, specificity is
The results show that h-D-glucosidase is a useful achieved by combining five enzymatic reactions and
marker enzyme for the differentiation of C. para- one reaction of unknown nature. The fingerprint
psilosis from other medically important yeasts on a resulting from the combined positive and negative
membrane filter. Within the genus Candida, the spe- reactions for C. parapsilosis is different from that of
cificity of this reaction for C. parapsilosis is fair. the 11 other common medical yeasts tested. Unlike
Cross-reaction with roughly 10% of C. albicans and other experimental conditions, the present ones are not
C. glabrata strains appears unacceptable in view of suitable for the differentiation of C. albicans from C.
the much higher prevalence of these species in clinical dubliniensis, as only 9% of C. albicans strains were
practice relative to that of C. parapsilosis. positive, while no reaction was observed for a strain of
When other non-Candida yeasts are also taken into C. dubliniensis.
account, specificity for C. parapsilosis is low, as a The method has the advantage of combining iso-
significant number of strains of the four species tested lation and identification and, hence, avoiding the need
also reacted. Smitka and Jackson (1989) already noted for a lengthy preliminary culturing step. In addition,
that h-D-glucosidase is not 100% specific for C. owing to a thorough optimization of the conditions forT.G.M. Bauters et al. / Journal of Microbiological Methods 53 (2003) 11–15 15
fluorescence development, with membrane filtration for rapid presumptive differentiation of four Candida species. J.
being a key factor, the test can be carried out at the Clin. Microbiol. 37, 1499 – 1502.
Bobey, D.G., Ederer, G.M., 1981. Rapid detection of yeast enzymes
microcolony level (Bauters et al, 1999). As a result, the by using 4-methylumbelliferyl substrates. J. Clin. Microbiol. 13,
method only takes 9– 11 h versus up to >48 h for that 393 – 394.
of Smitka and Jackson (1989), which requires prelimi- Moran, P., Sullivan, D., Coleman, D., 2002. Emergence of non-
nary isolation and culturing prior to the actual fluo- Candida albicans Candida species as pathogens. In: Calderone,
R.A. (Ed.), Candida and Candidiasis. ASM Press, Washington,
rescence assay as well as instrumental reading as a
DC, USA, pp. 37 – 53.
function of time. Pinjon, D., Sullivan, D., Salkin, I., Shanley, D., Coleman, D., 1998.
Simple, inexpensive, reliable method for differentiation of Can-
dida dubliniensis from Candida albicans. J. Clin. Microbiol. 36,
Acknowledgements 2093 – 2095.
Smitka, C.M., Jackson, S.G., 1989. Rapid fluorogenic assay for
differentiation of the Candida parapsilosis group from other
We are grateful to J. Meis (Regional Laboratory for
Candida spp. J. Clin. Microbiol. 27, 203 – 206.
Public Health, Nijmegen) and P. Verweij (University Sullivan, D., Coleman, D., 1998. Candida dubliniensis: charac-
Hospital, Nijmegen, The Netherlands) for providing teristics and identification. J. Clin. Microbiol. 36, 329 – 334.
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nicity, clinical manifestations, and antimicrobial susceptibility.
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References
Bauters, T.G.M., Peleman, R., Moerman, M., Vermeersch, H., De
Looze, D., Noens, L., Nelis, H.J., 1999. Membrane filtration testYou can also read
