Comparison of RAPD and ID 32C system for the identification of Candida isolates

Laura Baires-Varguez1, 2, Alejandro Cruz-García1, 2, Lourdes Villa-Tanaka2, Luis

Alberto Gaitán-Cepeda1, Luis Octavio Sánchez-Vargas1, Guillermo Quindós3 and

César Hernández-Rodríguez2*

1Facultad de Odontología, Universidad Nacional Autónoma de México2Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto

Politécnico Nacional, México, D.F. México3Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y

Odontología, Universidad del País Vasco-Euskal Herriko Unibersitatea, Bilbao, España.

*Corresponding author. Mailing address: Laura Baires-Varguez and César Hernández-

Rodríguez; Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas,

Instituto Politécnico Nacional, Apartado Postal CON 174. México, D.F. CP. 06400.

México. Phone and fax: (52 55) 57 29 62 09.

E-mail: baireslaura@yahoo.com.mx

Abstract

Numerous methods developed to differentiate strains of Candida species includes test kits,

such as the API 20C or ID 32C yeast identification systems, several studies have proposed

that randomly amplified polymorphic DNA (RAPD) analysis could be directly used as an

easy and secure tool for the identification of several pathogenic species.

We assessed the sensitivity of the molecular test (RAPD with the primer OPE-18) and

compared it to the biochemical test (IDE 32 C AUX system). Additionally identification of

C. albicans was compared with the biological test (germ tube test and chlamydospore

production), to determine its sensitivity and specificity, as well as its predictive values,

were performed. A total of 92 isolates (20 C. albicans, 14 Candida glabrata, 15 Candida

tropicalis, 11 Candida lusitaniae, 10 Candida guillermondii, 5 Candida parapsilosis, 7

Candida krusei, 1 Candida pelliculosa, 2 Candida rugosa and 7 Candida kefyr) obtained

from different clinical sites were analyzed. In addition, 9 culture collection strains were

included.

The RAPD-PCR patterns obtained with the primer OPE-18 for the identification of clinical

isolates were consistent, and the different independent assays revealed reproducibility of

the band patterns. The RAPD-PCR with the OPE-18 primer is a very specific and sensitive

method for the identification of important pathogenic Candida species included C.

glabrata, C. guilliermondii, C. tropicalis, C. pelicullosa, C. albicans, C. krusei, and C.

lusitaniae.

Introduction

Candida species have become an important cause of nosocomial infection, [1] Candida

albicans remains the most common cause of fungemia and disseminated candidiasis, other

Candida species are not uncommon [2, 3]. Thus, an early and accurate diagnosis of an

invasive fungal infection is critical for timely, appropriate management [4].

Numerous methods developed to differentiate strains of Candida species includes test kits,

such as the API 20C or ID 32C yeast identification systems [5-10], these can require

several days, even after the yeast has been recovered on fungal medium. Subjective scoring

inherent variations in colony morphologies and phenotypes among isolates within a species

of Candida, and the ability of some clinical isolates to grow in vitro are also problematic

[11]. Additionaly an increasing number of C. albicans isolates from AIDS patients are

atypical and cannot be reliably identified with several commercial yeast identification kits.

Molecular methods have been principally used to identify Candida isolates and to delineate

subgroups within a species for epidemiological purposes [2, 12, 13]. Several studies have

proposed that randomly amplified polymorphic DNA (RAPD) analysis could be directly

used as an easy and secure tool for the identification of several pathogenic species,

including Saccharomyces spp. [14], Penicillium spp. [15] and Candida spp [16].

Distinctive RAPD patterns have been described for identification of several species of

Candida [5, 16-18]. Polimerase Chain Reaction (PCR) methods are particulary promising

because of their simplicity, specificity, and sensitivity. However, current limitations exist

for routine use of the short primers in these RAPD profiles for clinical diagnosis [5].

The objective of this work was to analyze and to compare a RAPD-PCR assay with ID 32C

AUX kit and other biological test for the identification of different Candida species isolated

from clinical specimens.

Materials and methods

Candida isolates were obtained from four microbiological laboratories at the cities of

Mexico DF, Guadalajara, Monterrey and Guanajuato in Mexico.

A total of 92 isolates (20 C. albicans, 14 Candida glabrata, 15 Candida tropicalis, 11

Candida lusitaniae, 10 Candida guillermondii, 5 Candida parapsilosis, 7 Candida krusei, 1

Candida pelliculosa, 2 Candida rugosa and 7 Candida kefyr) obtained from different

clinical sites were analyzed. In addition, 9 culture collection strains were included.

All yeasts were identified by germ tube test, chlamydospore production and morphology on

cornmeal agar (Difco, USA) and Lee’s medium. Assimilation profiles were determined by

ID 32C test (bioMérieux, France). For DNA extraction, yeasts were grown on Sabouraud

dextrose agar plates (Difco) at 37°C for 24 to 48 h. A single colony was subcultured

overnight on YPD broth (1% yeast extract, 2% peptone, 2% dextrose) at 37°C and 200 rpm

agitation.

DNA was extracted from all yeast species by DNeasy isolation kit (Qiagen, USA). This kit

facilitates the rapid recovery of sufficient DNA for PCR amplification and allows multiple

samples to be extracted in parallel. DNA concentrations and A260/A280 ratios were

determined spectrophotometrically with o spectrophotometer (Lambda 1A; Perkin Elmer,

USA). An A260/A280 ratio of 1.8 to 2.1 was considered acceptable. The RAPD profiles

were obtained with primer OPE-18 (5’- GGACTGCAGA-3’) (Gibco BRL, USA).

RAPD analysis was performed by a previously described method [18] with minor

modifications. Reaction mixture contained: 1 l (10 ng/l) of genomic DNA, 1 l OPE-18

primer, 2 l of a deoxynucleotide triphosphate mixture (0.5 l each dATP, dCTP, dGPT

and dTTP), 1 l (2mM) MgCL2, 0.24 l (1.2 U) of Taq DNA polymerase in the PCR buffer

provided by the manufacturer (Gibco BRL). Amplification consisted of 38 cycles, each of

1 min at 94 °C, 1 min at 36°C and 2 min at 72 °C.

A 5 to 10 l sample of each PCR product was analyzed by electrophoresis in 1.2% (wt/vol)

agarose (Gibco BRL) gel slabs (14 cm by 10 cm by 6mm) with tris-acetate buffer (1XTAE;

0.04 M tris-acetate pH 8.4, 1 mM EDTA) at 80 V for 2 to 3 h. Gels were stained with 0.5

g of ethidium bromide per ml of deionized water for 20 min, followed by a 30 min wash

in deionized water. DNA bands confirming a positive PCR were visualized with a UV

transiluminator, photographed and documented with an Eagle Eye System (Strategene,

USA). The RAPD patterns obtained were analyzed by the Sigma Gel version 1.0

(JandelScientific, USA).

Results and discussion

We assessed the sensitivity of the molecular test and compared it to the biochemical test

(IDE 32 C AUX system) that is routinely used and has been amply validated. Additionally

identification of C. albicans was compared with the biological test (germ tube test and

chlamydospore production), to determine its sensitivity and specificity, as well as its

predictive values, were performed.

The overall clinical isolates were identified according to species usin genomic DNA

recently extracted by means of the commercial Dneasy kit for the RAPD-PCR

amplification test. We found that a commercial extraction kit (DNeasy) was an effective

and simple method for extraction DNA from pure yeast cultures within 30 min to

overnight. The only step used for DNA extraction with the kit was heating of yeast cells

suspensions in the lyses solution in a thermal cycler, eliminating the use of phenol-

chloroform and alcohol for DNA purification and precipitation, respectively. However, for

extraction of yeast DNA from cultures, we found that a prior step of lysing cells with

proteinase K followed by lyticase digestion of the yeast cell walls was necessary to obtain

good results [2].

Figure 1 shows representative RAPD-PCR products from each Candida species using

reference strains. This report revealed that the use of RAPD-PCR with the primer OPE-18

described by Lockhart [18] is very discriminatory to identify the most frequently

encountered species of yeast in clinical specimens. Consistent RAPD patterns were

obtained using OPE-18 oligonucleotide.

Table 1 depicts the molecular weights of the monomorphic RAPD bands considered for the

identification of the different Candida species.

RAPD methodology is rapid, accurate, reliable and simple for the early identification of

Candida isolates [5]. Identification of the fungal species is essential for the appropriate

clinical decision making concerning both the significance of a particular isolate and the

dosage and duration of antifungal therapy [19]. In addition, the use of RAPD fingerprints

can help in the study of epidemiology of nosocomial fungal infections by having the ability

to delineate among strains of the various Candida species evaluated [5].

As shown in Fig. 2, the RAPD-PCR patterns obtained with the primer OPE-18 for the

identification of clinical isolates were consistent, and the different independent assays

revealed reproducibility of the band patterns. Besides, specific amplifications for DNA

templates corresponding to each species were generated. The analysis of these bands

reveled profiles highly consistent. However, significant differences in the sizes (bp) of

bands were reported previously by Bautista-Muñoz [16] with the same primer (OPE-18)

and other primers. Moreover, we found a profile specific and sensitivity for C. pelliculosa

and a profile sensitivity for C. rugosa, but there was not a profile capable to differentiate C.

albicans and C. dubliniensis.

PCR assays with several other target sequences have been reported in the literature [20, 21].

However; several PCR assays based on amplification of the rDNA [22] and mitocondrial

DNA [23] have been recently reported. Species identification usually involves further

manipulation of the amplified products, using restriction enzyme digestion [24, 25],

radioactive or enzyme-labeled probes [26-28] or DNA sequencing [4, 29].

Sensitivity of RAPD-PCR, in our study, for the total of isolates was set al 0.91%; 84 of the

92 Candida isolates were correctly identified. The results of this study reinforce the value

of previously described RAPD analysis procedures for the identification of Candida species

[16].

Table 2 depicts the results and the differences obtained in species identification as well as

from the biological test. Our study and those of others reported previously [5, 6, 10, 17, 18,

30] have shown that RAPD methods performed with different oligonucleotides basically

generated consistent patterns, with several shared fragments unique to each species.

RAPD-PCR depicted 100% sensitivity and specificity for the identification of C. glabrata,

C. guilliermondii, C. tropicalis, and C. pelicullosa isolates.

For C. albicans, C. krusei, and C. lusitaniae, sensitivity was of the 100%, but specificity

was decreased, being of 0.96, 0.98 and 0.97%, respectively.

In contrast, for C. kefyr and C. parapsilosis, specificity was of 100%, but sensitivity was

smaller, 0.87 and 0.62%, respectively.

RAPD profiles are highly consistent due to the low degree of diversity and primary clonal

nature in populations of several pathogenic yeasts, including various species of Candida

[18, 31]. However, unfortunately a few reports [16] have been described the intraspecific

diversity and reproductive capabilities of the Candida species. All these data suggest that

the major monomorphic bands obtained by RAPD analysis are useful for the differentiation

of pathogenic Candida species.

RAPD fingerprints are species specific and simple enough to obtain the identification

without computer-assisted analysis [5].

C. rugosa was identified by RAPD-PCR in two isolates, but only one corresponded to this

species.

C. dubliniensis and C. colicullosa were not identified at all by RAPD-PCR.

The utility of RAPD profiles for the primary identification and typing of yeast and other

fungi yeast will require further evaluation. Even if different laboratories can adopt the

method with ease, the cost of the assay increases substantially if several RAPD primers are

needed for species identification [17].

The biological test based on formation of germ tubes and chlamydospores have been

widely used as C. albicans species-specific confirming test. Germ tube formation for this

species depicted a sensitivity and specificity of 0.88 and 0.99%, respectively; whereas, for

chlamydospore formation, sensitivity and specificity were 0.76 and 0.97%, respectively,

being the probability of true negative cases higher for both test.

Probability of predictive values was lower the biological test than for RAPD-PCR in the

identification of C. albicans, being high for the remainder species with the formed test

(Table 2)

In summary, the RAPD-PCR with the OPE-18 primer is a very specific and sensitive

method for the identification of important pathogenic Candida species included C.

glabrata, C. guilliermondii, C. tropicalis, C. pelicullosa, C. albicans, C. krusei, and C.

lusitaniae.

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Table 1. RAPD-PCR diagnostic monomorphic bands

Species Sizes (bp) of RAPD monomorphic

bands

C. albicans 2,219; 1,391; 994

C. glabrata 1,049; 949

C. guillermondii 2,864; 1,638; 935; 835; 585

C. kefyr 1,638; 1,508; 917; 667

C. krusei 3,052; 1,505; 961

C. lusitaniae 2,101; 1,982; 1,055; 836

C. parapsilosis 1,232; 759

C. rugosa 1,330; 1,036; 775; 498

C. tropicalis 1,839; 1,638; 810; 706; 602

Table 2. Comparison of clinical yeast isolates identifications made by IDE 32C, RAPD/PCR, and the biological test based on

formation of germ tubes and chlamydospores

Species No. of clinical

isolates tested

IDE 32C AUX RAPD/PCR Germ tube Clamidospores

C. albicans 17 17 20 15 13

C. dubliniensis 2 2 0 0 2

C. glabrata 14 14 14 0 0

C. guilliermondii 10 10 10 0 0

C. kefyr 8 8 7 0 0

C. krusei 6 6 7 0 0

C. lusitaniae 9 9 11 0 0

C. parapsilosis 8 8 5 0 0

C. tropicalis 15 15 15 7 2

C. rugosa 1 1 2 0 0

C. pelicullosa 1 1 1 0 0

C. colicullosa 1 1 0 0 0

TOTAL 92 92 92 22 17

Table 3. Sensitivity and specificity of RAPD/PCR for the identification of species of Candida and the biological test for

identification of Candida albicansSpecies No. RAPD/PCR Primer OPE-18 Germ tube Clamidospores

S E VP + VP - S E VP + VP - S E VP + VP -

C. albicans 17 100 0.96 0.84 0.99 0.88 0.91 0.67 0.97 0.76 0.97 0.86 0.95

C. dubliniensis 2 0 0 NA NA

C. glabrata 14 100 100 NA NA

C. guillermondii 10 100 100 NA NA

C. kefyr 8 0.87 100 100 0.99

C. krusei 6 100 0.98 0.99 100

C. lusitaniae 9 100 0.97 0.99 100

C. parapsilosis 8 0.62 100 100 0.96

C. tropicalis 15 100 100 NA NA

C. rugosa 1 100 50 NA NA

C. pelicullosa 1 100 100 NA NA

C. colicullosa 1 0 0 NA NA

S=Sensitivity P≥ 0.05 E=Specificity P≥ 0.05

PV += Predictive values positive P≥ 0.05 PV -= Predictive values negative P≥ 0.05 NA=No aplicable

Figure 1. RAPD-PCR performed with genomic DNA (primer OPE-18) from culture collection strains of Candida. The different

independent assays revealed reproducibility of the band patterns

Figure 2. The overall clinical isolates were identified according to species usin genomic DNA (primer OPE-18). RAPD/PCR

patterns of different Candida species were very consistent. The figure shows amplicons from each of individual clinical isolates.

Lanes 51, 60, 62 and 64 were eliminated. The first lane in each serial corresponding to DNA ladder.