identification of clinically relevant yeasts by...

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Journal of Microbiological Methods 56 (2004) 201–211

Identification of clinically relevant yeasts by PCR/RFLP

Anja Trosta,b, Barbara Grafa, Jan Euckerb, Orhan Sezerb, Kurt Possingerb,Ulf B. Gobela, Thomas Adama,*

a Institute for Microbiology and Hygiene, Medical Faculty of Humboldt University, Charite, Dorotheenstr. 96, 10117 Berlin, GermanybDepartment of Medical Oncology/Hematology, Charite, Humboldt-University, 10117 Berlin, Germany

Received 23 May 2003; received in revised form 10 October 2003; accepted 21 October 2003

Abstract

For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of

appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most

diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or

low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of

clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we

describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of

PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida

species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.

D 2003 Elsevier B.V. All rights reserved.

Keywords: PCR products; Yeasts; Candida species

1. Introduction species has been observed, with an increase of non-

The incidence of invasive fungal infections has

increased in recent years, being a major cause of

morbidity and mortality in immunocompromised

patients, such as recipients of bone marrow trans-

plants, patients with hematological malignancies with

or without chemotherapy, and AIDS patients (Toscano

and Jarvis, 1999; Martin et al., 2003). Candida

albicans is the most common pathogenic Candida

species. However, a shift in the spectrum of Candida

0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.mimet.2003.10.007

* Corresponding author. Tel.: +49-30-450-524229; fax: +49-30-

450-524908.

E-mail address: [email protected] (T. Adam).

albicans Candida species being found in clinical

isolates (Viscoli et al., 1999). Increasing incidence

of candidemias caused by Candida parapsilosis and

Candida glabrata (Malani et al., 2001), Candida

tropicalis, Candida krusei, Candida guillermondii or

Candida lusitaniae (Kao et al., 1999) has been de-

scribed. Candida dubliniensis was found to be asso-

ciated with HIV infection and AIDS (Sullivan et al.,

1995). However, recent reports on C. dubliniensis

infections in HIV-negative patients, including funge-

mias, make identification of this pathogen crucial for

routine laboratories (Meis et al., 1999; Brandt et al.,

2000; Marriott et al., 2001; Fotedar and Al Hedaithy,

2003; Yang et al., 2003). Given the high lethality of

invasive Candida infections and different sensitivities

A. Trost et al. / Journal of Microbiological Methods 56 (2004) 201–211202

to antimycotics of the various Candida species, early

and accurate identification of Candida species is

critical for appropriate choice of antifungal therapy.

Traditional phenotypic methods of identification of

fungi used in routine laboratory settings are time-

consuming and may lead to ambiguous results.

A number of procedures using PCR amplification

techniques have been suggested for identification of

Candida species. The ribosomal genes are popular

targets for PCRbased systems for detection and iden-

tification of fungal pathogens (Kappe et al., 1996;

Einsele et al., 1997). However, identification of yeast

on the species level is difficult to achieve due to

limited variation of common yeast species on the 18S

rDNA level; in the genus Aspergillus, there is even

less 18S rDNA sequence variability between species.

Therefore, 18S rDNA-based Aspergillus probes are

not species-specific (Einsele et al., 1997; Kappe et al.,

1998; Reiss et al., 1998).

The internal transcribed spacer regions ITS1 and

ITS2 are highly variable sequences that have been

used for identification of fungi on the species level in

different formats (Fujita et al., 1995; Elie et al., 1998;

Reiss et al., 1998; Martin et al., 2000; Lindsley et al.,

2001; Selvarangan et al., 2002). Identification of the

amplicon is an important issue in all PCR-based

systems. Hybridization with species-specific DNA

probes, direct sequencing of the PCR product (White

et al., 1990) or precise determination of lengths of

PCR products (Turenne et al., 1999; Chen et al., 2000;

Chen et al., 2001; De Baere et al., 2002) are time-

consuming methods or require expensive equipment

not readily available in many diagnostic laboratories.

Analysis of restriction fragment length polymor-

phisms (RFLP) of PCR products is a rapid and simple

technique that requires only standard instrumental

equipment, and provides unambiguous results. This

technique is therefore easily transferable to most

diagnostic laboratories. We have recently established

a PCR/RFLP system for rapid and simple differenti-

ation between C. albicans and C. dubliniensis (man-

uscript submitted). The PCR/RFLP system described

here identifies 12 clinically significant yeast species

by PCR amplification of the ITS1 and ITS2 regions

and subsequent species-specific enzymatic cleavage

with the endonuclease MwoI. Using a second restric-

tion digest with BslI, all 16 species of the test panel

could be identified unequivocally.

2. Methods

2.1. Yeast strains

For studies of intra-species variability, we used

clinical strains of C. glabrata (32 isolates), C. tropi-

calis (10), C. krusei (7), Candida kefyr (7), C. para-

psilosis (3) and one strain each of C. guillermondii,

Candida inconspicua, Candida norvegensis and Sac-

charomyces cerevisiae. These isolates were character-

ized in our diagnostic laboratory using phenotypical

methods including formation of chlamydospores on

rice extract Tween 80 agar (Kreger-Van Rij, 1984),

characteristic growth on CHROMagar (Mast Diag-

nostics), and growth at 45 jC (Pinjon et al., 1998). All

other strains used in this study were from international

culture collections (ATCC, American Type Culture

Collection, Manassas, VA, USA; DSMZ, Deutsche

Sammlung von Mikroorganismen und Zellkulturen,

Braunschweig, Germany; CBS, Centraalbureau voor

Schimmelcultures, Utrecht, The Netherlands): C. albi-

cans: ATCC 90028, C. dubliniensis: CBS 7987; C.

glabrata: ATCC 90030; C. guillermondii: DSMZ

6381; C. inconspicua: DSMZ 70631; C. kefyr: DSMZ

11954; C. krusei: ATCC 6258; C. lusitaniae: DSMZ

70102; Candida magnoliae: ATCC 201379; Candida

membranaefaciens: ATCC 201377; C. parapsilosis:

ATCC 90018;C. tropicalis: DSMZ 5991;C. norvegen-

sis: DSMZ 70760; Cryptococcus neoformans: ATCC

90112; Trichosporon mucoides: ATCC 201383; S.

cerevisiae: ATCC 9763.

2.2. Culture conditions and DNA extraction

Strains were cultivated on solid Sabouraud agar

plates for 3–4 days at 30 jC. LB medium (10 ml) was

inoculated and grown in 50 ml tubes (Falcon, Becton

Dickinson, Cockeysville, NJ, USA) at room tempera-

ture with shaking over night.

After centrifugation at 2268� g for 10 min, the

supernatant was removed and the pellet was washed by

adding 20 ml of washing buffer (150 mM NaCl, 10

mM Tris–HCl, 0.5 mM EDTA, pH 8.0) at room

temperature. Again, the suspension was centrifuged

at 2268� g for 10 min and the supernatant removed.

For preparation of DNA (method modified from Mull-

er et al. (1998)), the resulting pellet was resuspended in

500 Al TE buffer (10 mM Tris–HCl, pH 7.6, 1 mM

Table 1

Fungal species with complete sequences of the primer binding sites

for PCR1 available in GenBank/EMBL

Organism GenBank accession numbers

Binding site

of primer

Binding site

of primer 3

1/1a

(18S rDNA)

(25/28S rDNA)

Yeast species

Candida albicans af331936 l28817

Candida dubliniensis aj010332 af405231

Candida glabrata x51831 y15467

Candida guillermondii ab013587 –

Candida kefyr y15476

Candida krusei m55528 –

Candida tropicalis m55527 –

Saccharomyces cerevisiae u53879

(1 mismatch)

u53879

Aspergillus species

Aspergillus avenaceus ab008395 af104446

Aspergillus flavus x78537,

d63696

af109341

Aspergillus fumigatus m60300 af078889,

u28461-3

Aspergillus nidulans ab008403 –

Aspergillus niger d63697 af108474,

af109343/4

Aspergillus nomius ab008404 af338617

Aspergillus ochraceus ab002068 –

Aspergillus oryzae d63698 –

Aspergillus parasiticus d63699 af027862

Aspergillus penicilloides ab002078 –

Aspergillus sojae d63700 –

Aspergillus tamarii d63701 af004929

Aspergillus terreus ab008409 af109339

Aspergillus ustus ab008410 –

A. Trost et al. / Journal of Microbiological Methods 56 (2004) 201–211 203

EDTA, pH 8.0) and transferred to a 2-ml screw cap

tube filled with 800 mg of glass beads (G-8772,

Sigma-Aldrich, Steinheim, Germany), 600 Al of phe-nol was added. For cell disruption the tubes were

shaken three times for 30 s in a Fast-Prep Shaker

(FP120, Bio101, dianova, Hamburg, Germany) at

speed 5.5 and chilled on ice after each session. The

tubes were then centrifuged at 4 jC and 15,366� g on

a cooled tabletop centrifuge for 15 min, and the liquid

phase and the interphase, containing the DNA, were

transferred to a 2-ml tube. DNA was further extracted

by phenol–chloroform treatment and ethanol-precipi-

tated as described (Sambrook and Russel, 2001).

2.3. Sequence information, primer design and selec-

tion of restriction enzymes

Oligonucleotide primers for polymerase chain reac-

tions (PCR) were obtained from MWG Biotech

(Ebersberg, Germany). Primers for PCR1 were

designed to amplify the internal transcribed spacer

regions 1 (ITS1) and 2 (ITS2), including the 5.8S

rDNA of the most frequent fungal pathogens in

humans (Fig. 1). In addition to the sequences of a

number of yeast species, the sequences of Aspergillus

species were taken into account, considering potential

future use of the method for identification of patho-

genic Aspergillus species. Published sequences avail-

able in the GenBank/EMBL database were used for

alignments of the considered species. Primers 1 and 1a

bind to 18S rDNA, primer 3 to 25/28S rDNA, repre-

senting highly conserved regions of DNA. Primers 1

(5V-GTCAAACTTGGTCATTTA-3V) and 1a (5V-GTCAAACCCGGTCATTTA-3V) were chosen as for-

ward primers with sequence identity to the available

sequences of the pathogens listed in Table 1. Primer 1a

differs in two nucleotides from primer 1 in order to

Fig. 1. Organization of the fungal ribosomal genes. Primer target areas used

ITS2 are indicated. Primer 1: forward primer binding to 3V-end of 18S rDN

18S rDNA of Aspergillus species. Primer 3: reverse primer binding to 5Vreverse primer binding to 5V-end of 5.8S rDNA of selected yeast species.

include relevant Aspergillus species for future appli-

cations of the method to clinically relevant molds.

Primer 3 (5V-TTCTTTTCCTCCGCTTATTGA-3V) is

in the amplification of internal transcribed spacer regions ITS1 and

A of yeast species. Primer 1a: forward primer binding to 3V-end of

-end of 25/28S rDNA of yeast and Aspergillus species. Primer 2:


identical to the respective 25/28S rDNA sequences of

all fungal species listed in Table 1. Primer 2 (5V-CCAAGAGATCCA/GTTGTT-3V) was used for PCR

amplification of ITS1 together with primers 1 and 1a

(PCR2). The primers were chosen as not to amplify

human DNA or DNA of relevant nonfungal human

pathogens.

Restriction enzymes were selected as to differenti-

ate a maximum of the PCR1 products of the 16 yeast

species analyzed here. Thus, the selected enzymes

were required to have at least one cleavage site in

the amplicon of each species, resulting preferably in

DNA fragments with a minimum length of about 80 bp

that are easily visible in conventional agarose gels.

2.4. DNA sequencing

We used the novel USERk (New England Biolabs,

Frankfurt, Germany) system for cloning of PCR1

products (Fig. 1) from C. parapsilosis (ATCC

90018) and Candida membranaefaciens (ATCC

201377) in pNEB205A according to the instructions

of the manufacturer. Using a ThermoSequenase primer

cycle sequencing kit (Amersham, Freiburg, Germany),

nucleotide sequences of three independent clones,

respectively, were determined in both directions on a

LiCor 4000L sequencer.

2.5. PCR

The ITS region primers make use of conserved

regions of the 18S (primer 1/1a), 5.8S (primer 2) and

the 25/28S rRNA (primer 3) genes to amplify the

intervening 5.8S gene and the noncoding Internal

Transcribed Spacer regions ITS1 and ITS2 (PCR1,

primers 1/1a, 3), or only ITS1 (PCR2, primers 1, 2)

(Fig. 1). Optimal specificity was achieved in a total

reaction volume of 100 Al consisting of 1� of PCR

buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl), 1.5

mM MgCl2, 200 nM primer, 50 AM dNTPs, 1 U

Ampli-Taq DNA polymerase and water (ad 100 Al).Thirty-four cycles of amplification were performed on

a thermocycler (DNA Thermal Cycler 480, Perkin

Elmer Cetus, Norwalk, USA) after initial denaturation

of DNA at 94 jC for 3 min. Each cycle consisted of a

denaturation step at 94 jC for 30 s, an annealing step at

50 jC for 30 s and an extension step at 72 jC for 1

min, with a final extension at 72 jC for 10 min

following the last cycle. The PCR products were

analysed by electrophoresis on 2% agarose gels and

stained with ethidium bromide. Gene Ruler DNA

ladder Mix (SM0331, MBI Fermentas, St. Leon-Rot,

Germany) was used as DNA marker. PCR products

were purified using the QIAquick PCR Purification kit

(Qiagen, Hilden, Germany) according to the instruc-

tions of the manufacturer. PCR products were eluted in

40 Al of elution buffer included in the kit.

2.6. Restriction digests of PCR1 products

Restriction digests were set up with 12 Al of puri-fied or nonpurified PCR1 product and 1 U of the

respective enzyme (MwoI or BslI, New England Biol-

abs) and incubated for 2 h at 60 jC for the MwoI

digests and 55 jC for the BslI digests.

Restriction fragments were separated by electro-

phoresis in 2% agarose gels. Gene Ruler DNA Ladder

Mix (SM0331, MBI Fermentas) was used as DNA

marker. The gels were stained with ethidium bromide

and analysed with an Eagle Eye Illuminator (Strata-

gene, Heidelberg, Germany).

3. Results

3.1. Sequence analyses

In order to design PCR primers for a wide range of

fungal species, EMBL/GenBank entries containing

intended target sequences were used for sequence

alignments. Conserved target sequences for PCR pri-

mers were chosen to enable amplification of a broad

range of clinically relevant fungi. On the other hand,

the amplicon should be large enough and contain

variable sequences that were shown to allow identifi-

cation of fungal species. We therefore decided to

amplify the complete ITS1 and ITS2 regions using

PCR primers targeting conserved sequences of adja-

cent upstream 18S and downstream 25/28S rRNA

genes (Fig. 1). Database entries containing complete

sequences of primer targets are given in Table 1. With

the exception of S. cerevisiae (one mismatch in the 5V-region of primer 1), we found complete sequence

identity of primer 1 with 18S rDNA sequences of

yeasts, and of primer 1a with the respective rDNA

sequences of molds.


In some species, complete sequence information of

the primer binding regions was not available. There-

fore, additional database entries containing incom-

plete sequences for the primer binding regions were

used (data not shown).

We did an exhaustive analysis of databank entries

with respect to calculated fragment lengths of the

PCR1 products of commercially available restriction

enzymes. The criteria for the selection of an enzyme to

be evaluated in experiments were the presence of at

least one cleavage site for every species of our panel

and the generation of a great variety of fragment length

patterns that should easily be distinguishable in con-

ventional agarose gels. However, since complete se-

quence information was not available for all species of

our panel of strains, we could not predict the complete

set of restriction profiles. According to these criteria,

we chose the restriction enzyme MwoI. Predicted

fragment lengths of MwoI-digested PCR1 products

are given in Table 2. In addition, we determined the

nucleotide sequences of PCR1 products from C. para-

psilosis and C. membranaefaciens. These sequences

Table 2

GenBank/EMBL data bank entries used for calculation of PCR1 amplicon

Species GenBank accession no.

of data bank entries

used in this study

Calculate

of PCR1

(bp)

C. albicans af331936 586

af217609

aj249486

C. dubliniensis aj010332 589

aj249484

x99399

C. glabrata x51831 925

l47108

af218966

y15467

C. krusei m55528 560

l47113

(l11350)

C. tropicalis m55527 576

af287910

af218992

C. parapsilosis aj585347a 570

C. guillermondii ab013587 657

ab032176

af218996

C. membranaefaciens aj585348a 686

S. cerevisiae scl9634 891

a This study, note that these data bank entries do not include primer b

allowed us to verify our RFLP results with MwoI or

BslI, respectively. As given in Table 2 and Figs. 2–4,

the predicted fragment sizes match lengths of PCR1

and PCR2 products and RFLP patterns as seen in

agarose gels.

3.2. PCR1

As shown in Fig. 2, DNA from all yeast species

included in this study could be amplified with primers

1/1a and 3 (PCR1). PCR1 products were found to

reach from 480 bp (C. lusitaniae) to 929 bp (C.

glabrata) in length (Fig. 2; Table 2). In yeast species

with complete sequence information of PCR1, empir-

ical lengths of PCR products were as predicted from

sequence analyses (Fig. 2; Table 2).

3.3. MwoI digests of PCR1 products

We first tested the ability of MwoI to cut the PCR

products of all species of our panel, including species

with incomplete sequence information. Restriction

lengths and fragment sizes of MwoI or BslI fragments

d length

product

Calculated lengths

of PCR1–MwoI

fragments (bp)

Calculated lengths

of PCR1–BslI

fragments (bp)

261, 184, 141

325, 264

414, 174, 171, 86, 80

289, 134, 83, 49, 5

325, 154, 97 326, 187, 63

336, 146, 88 413, 94, 63

355, 302 356, 238, 63

387, 299 623, 63

343, 207, 173, 168

inding sites.

Fig. 2. Results obtained after PCR1 of 16 common yeast species with primers 1, 1a and 3. PCR products were electrophoretically separated

using a 2% agarose gel.


digests proved the presence of at least one MwoI

cleavage site in all yeast species examined in our

study (data not shown; see Fig. 3). We next studied

Fig. 3. RFLP patterns of the PCR1 products shown in Fig. 2 after dige

fragments on a 2% agarose gel. Unequivocal identification of 12 yeast spec

tropicalis and C. parapsilosis or C. guillermondii and C. membranaefacie

conditions of agarose gel electrophoresis for docu-

mentation and analysis of restriction fragments. We

found optimal separation of DNA fragments using 2%

stion with endonuclease MwoI and electrophoretical separation of

ies by distinct patterns; two pairs of species show similar patterns: C.

ns, respectively.

Fig. 4. PCR1 products and their RFLP patterns of C. guillermondii and C. membranaefaciens (A) or C. tropicalis and C. parapsilosis (B) after

restriction digest with BslI. This restriction allows unequivocal differentiation of the remaining four species. (C) Product lengths of PCR2

distinguish C. guillermondii from C. membranaefaciens. (D) The effect of purification on electrophoretic mobility of PCR1–MwoI digestion

products of C. glabrata. Nonpurified digest migrates slightly slower during agarose gel electrophoresis.


agarose gels in 0.5� TAE buffer (data not shown).

RFLP analysis in agarose gels was simple and repro-

ducible. The band patterns of the 16 yeast species

resulted in 14 easily recognizable patterns with two

patterns representing two strains each (Fig. 3). While

12 yeast species were characterized by distinct restric-

tion patterns, C. parapsilosis could not easily be

distinguished from C. tropicalis, and C. membranae-


faciens could not be discriminated from C. guiller-

mondii on the basis of MwoI restriction profiles (Fig.

3, lanes 5 and 6 or 10 and 11, respectively). Though

the two pairs of similar restriction profiles showed

some minor differences in fragment lengths, these

differences were not considered reliable markers un-

der routine conditions.

3.4. BslI digests of PCR1 products

In order to unequivocally distinguish C. parapsi-

losis from C. tropicalis and C. membranaefaciens

from C. guillermondii, we analysed published sequen-

ces of these species and chose restriction enzyme BslI

for this purpose. As shown in Fig. 4A and B, BslI

fragment patterns clearly distinguished C. parapsilo-

sis from C. tropicalis and C. membranaefaciens from

C. guillermondii, respectively. RFLP patterns of C.

parapsilosis and C. membranaefaciens were con-

firmed by DNA sequencing of the PCR1 products

(Table 2).

3.5. PCR2 for differentiation between Candida

species with similar PCR1–MwoI patterns

We also performed a shorter PCR that amplified

only the ITS1 region (PCR2) to distinguish between

C. membranaefaciens and C. guillermondii. We found

that differences in PCR2 product lengths between

these Candida species could easily be detected in

conventional agarose gels (Fig. 4C). Calculated

lengths of PCR2 products are 267 bp for C. guiller-

mondii and 299 bp for C. membranaefaciens. PCR2 is

thus an alternative method to BslI-RFLP analysis of

PCR1 products to distinguish C. membranaefaciens

from C. guillermondii.

3.6. Effect of purification of restriction digests on

electrophoretic mobility

Precise analysis of predicted MwoI patterns indi-

cated that restriction fragments appeared slightly larger

than calculated from database entries. We hypothe-

sized that this could be due to factors that may attach to

DNA fragments during MwoI digestion of PCR1

products. We therefore compared electrophoretic mo-

bility of purified vs. nonpurifed PCR1–MwoI digests

from C. glabrata. As shown in Fig. 4D, the non-

purified digest migrates slightly slower than the puri-

fied digest.

3.7. Intra-species conservation of restriction sites

We next wanted to know whether MwoI and BslI

restriction sites are conserved among a limited

number of clinical yeast isolates. We therefore

studied a number of clinical isolates, identified using

classical phenotypic methods in our diagnostic lab-

oratory, to assess intra-species variability of MwoI or

BslI RFLP patterns, respectively. All 32 clinical C.

glabrata strains tested showed a single MwoI-RFLP

pattern identical to the reference strain (data not

shown). In addition, we studied PCR1–MwoI pat-

terns of C. tropicalis (10 strains tested), C. krusei

(7), C. kefyr (7), C. parapsilosis (3) and one strain

each of C. guillermondii, C. inconspicua, C. norve-

gensis and S. cerevisiae. The PCR1–MwoI patterns

of all these isolates were identical to the respective

MwoI patterns of reference strains (data not shown).

Similar results were obtained with PCR1–BslI

digests of 10 C. tropicalis, 3 C. parapsilosis and

one C. guillermondii isolates (data not shown). In a

recent study, we had shown that MwoI sites of the

PCR1 product were conserved in all 60 C. albicans

and 22 C. dubliniensis strains tested (in press).

Thus, we did not find intra-species variability of

PCR1–MwoI or PCR1–BslI restriction fragment

patterns among the clinical isolates tested indicating

that the MwoI and BslI sites used for characteriza-

tion of the PCR1 products generated in our system

are not situated in hot spots of extreme intra-species

variability.

4. Discussion

A PCR/RFLP system was developed for the

identification of clinically relevant yeast species.

PCR primers (modified from (White et al., 1990))

were designed for amplification of ITS1 and ITS2

regions of yeast and Aspergillus species of clinical

significance. For our method, PCR primers were

optimized in order to gain easily distinguishable

restriction fragments. For potential amplification of

a broad range of fungi including clinically relevant

yeasts and molds, positions of PCR primers were


chosen in conserved regions of 18S or 25/28S rRNA

genes, respectively, resulting in PCR products large

enough for optimal resolution of restriction frag-

ments on simple agarose gels. However, better res-

olution of band patterns can be achieved using

precast polyacrylamide gels. Since most fungi pos-

sess multicopy rDNA genes, choice of these genes as

targets for PCR primers makes use of natural ‘am-

plification’ of target sequences prior to PCR. Using

the described primers we could amplify DNA from

all 16 yeast species tested including Candida, Cryp-

tococcus, Saccharomyces and Trichosporon repre-

senting a broad range of clinically relevant yeasts.

The choice of restriction enzymes was also directed

by our aim to create a simple test with optimal

resolution of restriction profiles. Based on sequence

analyses of yeast sequences available in GenBank/

EMBL, the endonuclease MwoI was found to be the

most powerful enzyme for this purpose. MwoI

showed optimal discriminatory power, being able to

distinguish 12 yeast species and two pairs of species

with a single restriction digest. Unequivocal identi-

fication of these species was achieved including the

clear distinction between C. albicans and C. dublin-

iensis. BslI was found to discriminate the nondistin-

guishable species after restriction with MwoI. Using

this enzyme for an additional digest of the PCR1

product, all of the 16 yeast species studied here could

be identified. In addition, amplification of ITS1 alone

(PCR2) allowed differentiation of one of the two

pairs of Candida species that could not easily be

distinguished based on PCR1–MwoI restriction frag-

ment patterns (C. membranaefaciens/C. guillermon-

dii). The known variations in length of PCR products

from fungi spanning ITS1 and/or ITS2 regions have

successfully been used for unequivocal identification

of a large number of yeasts and molds (Turenne et

al., 1999; Chen et al., 2000; Chen et al., 2001; De

Baere et al., 2002). Using an amplicon spanning both

ITS1 and ITS2 regions, the simple approach de-

scribed here allows confirmation of PCR/RFLP

results with several described methods for identifica-

tion of fungi. In particular, methods based on size

variation of PCR products can be applied with slight

modification; in addition, a number of DNA probes

for identification of yeasts and molds targeting ITS1

or ITS2 regions can be used for confirmation of

PCR/RFLP results (Fujita et al., 1995; Elie et al.,

1998; Reiss et al., 1998; Lindsley et al., 2001).

Therefore, species not included in the panel exam-

ined here or emerging new fungal species that might

show equivocal results in the restriction assay should

easily be identified by the sequence information of

their PCR products.

Other RFLP-based assays have been described for

the presumptive identification of yeasts to the spe-

cies level. Maiwald et al. (1994) developed a method

on the basis of a PCR product of the 18S rDNA of

12 clinically relevant yeasts. Using six restriction

enzymes, eight species could be characterised lea-

ving two pairs of species with similar fragment

patterns. Williams et al. (1995) could clearly identify

eight yeast species using a combination of three

restriction enzymes for digestion of amplified ITS

regions ITS1 and ITS2. Esteve-Zarzoso et al. (1999)

identified 132 yeast species by profiling them with

information about the size of PCR products spanning

the ITS1 and ITS2 regions and restriction fragments

after digestion with three different enzymes. Vele-

graki et al. (1999) used four restriction enzymes for

the identification of the genera Aspergillus, Candida

and Cryptococcus. Species-specific patterns were

produced of four yeast species. However, a distinc-

tion between C. albicans and C. dubliniensis was

not described in either of those publications, while

other methods to discriminate these species are

limited to this purpose (Williams et al., 2001). Thus,

the simple PCR/RFLP system described here should

be instrumental in routine clinical laboratories for

fast and economic identification of clinically impor-

tant yeasts.

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