Genetic identification of Candida species in HIV-positive patients using the polymerase chain reaction and restriction fragment length polymorphism analysis of its DNA

Molecular and Cellular Probes (1999) 14, 401–406 Article No. mcpr.1999.0266, available online at http://www.idealibrary.com on

Genetic identification of Candida species in HIV-positive patients using the polymerase chain reaction and restriction fragment length polymorphism analysis of its DNA J. Irobi,1∗ A. Schoofs2 and H. Goossens3 1

Department of Biochemistry, University of Antwerp, UIA, Antwerp, 2Department of Dermatology, University Hospital Antwerp, UIA, Antwerp and 3Department of Microbiology, University Hospital Antwerp, UIA, Antwerp, Belgium (Received 2 July 1999, Accepted 12 August 1999)

The polymerase chain reaction was used to amplify a targeted region: an internal transcribed spacer region of the ribosomal DNA from 114 Candida isolates and 65 reference strains. Unique product sizes were obtained for Candida glabrata, C. guillermondii and C. inconspicua. Isolates of C. albicans, C. tropicalis, C. dubliniensis and C. krusei could be identified following restriction digestion of the PCR products. The methods proved to be both simple and reproducible and may offer potential advantages over phenotyping methods.  1999 Academic Press

KEYWORDS: identification of Candida species, polymerase chain reaction, restriction fragment length polymorphism, internal transcribed spacer region of the ribosomal DNA, bead beater, genetic variation.

INTRODUCTION

Candida species are responsible most commonly for chronic superficial infections of skin, nails and mucosal surfaces. Less frequently, they can disseminate and cause life-threatening systemic (or deep-seated) diseases, and these infections actually present the greatest challenge to clinicians. There are at least 166 species in the genus Candida,1 but only a small proportion of these are found in man, and of these only a handful pose clinical problems. Candida albicans is the most frequently isolated colonizing species and in most studies makes up 60–75% of the recovered yeast from sites of infection. Several non-albicans Candida species including C. dubliniensis, C. tropicalis, C. pseudotropicalis, C. krusei, C. guillermondii, C. parapsilosis and C. stellatoidea are known to be pathogenic in man or cause disease occasionally in

immunodeficient patients.2 Isolates of C. dubliniensis have been recovered mainly from the oral cavities of HIV-infected individuals and are most frequently implicated in cases of recurrent infection following antifungal drug treatment.2 Candida tropicalis was reported to be the most frequently isolated fungal pathogen for patients with hematologic malignancies.3 Candida parapsilosis has been linked to hyper alimentation and was the most frequent Candida species to cause intravenous drug use-related endocarditis4 encountered in otherwise apparently healthy women.5 Identification of the various species of these yeasts is a daunting problem. Traditional methods used for identification of clinical isolates of Candida species include morphological and biochemical analysis such as microscopic examination, germ tube test, chlamydospore formation on rice cream agar, API ID 32 (carbohydrate assimilation test)

∗ Author to whom all correspondence should be addressed at: Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Tel: +32 3 820 25-51/32 3 820 23-04; Fax: 32 3 820 26-63/32 3 820 25-41; E-mail: [email protected]

0890–8508/99/060401+06 $30.00/0

 1999 Academic Press

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and identification on CHROMagar medium. Thus, currently used methods are mainly based on phenotypic characteristics and can therefore lead to inconsistent results. For Instance, phenotypic switching of Candida species has been well documented;6,7 therefore genotype based approaches may show advantages over phenotypic methods. The aim of the present study was to evaluate a new polymerase chain reaction (PCR) based approach for the identification of seven species of Candida on the basis of size and variation of the rDNA internal spacer regions. The reliability of the technique was illustrated by the examination of a large number of both reference and clinical Candida isolates.

MATERIALS AND METHODS Isolates Sixty-five reference strains of Candida species provided by F. Odds (Janssen Research Foundation Beerse, Belgium) were included: C. albicans (J930505, 930509, J930510, J930817, J932262/1, J932571, J941019, J941389, B69976, B700283); C. dubliniensis (88/029, 89/014, 90/013, 90/015, 90/033, 90/033); C. glabrata (J930562/2, J950605, J931029/ 2, J931324, J931545, J940163(P), J930761, J950601, J950602, J950603); C. tropicalis (NCCLS 1, NCCLS15, NCCLS20, J930950, CDC49, J942105, J950226, J950587, J950588, J950589); C. krusei (J930433, J930435, J941366, J940838, J941805, J941366, J941838, J940208, J950607, J950606); C. inconspicua (SBS180, SBS990, SBS1735, SBS2833, SBS4258); and C. guilliermondii (J932047/2, J932070/2, J932726, B52446/4, B53007, B42092/2, J941180, J941267, SBS2083). Between December 1994 and September 1995, 130 oral rinses were collected after verbal consent from 130 HIV infected patients, comprising 35 seropositive individuals and 95 AIDS patients. These patients were attending the outpatient clinic of the Institute of Tropical Medicine, Antwerp, or hospitalized in the University Hospital Antwerp. Onehundred and fourteen Candida isolates were obtained from 81 of these patients. These isolates were identified by germ tube production and the AP1-20C system (bioMe`rieux, Chaibonniere du bain, France).8

DNA Preparation DNA was isolated from broth cultures. Yeast cells were grown in 10-ml medium composed of 5 g of Yeast extract powder, Oxoid L21, 20 g of D+ glucose,

Merck 8337, per 1000 ml distilled water overnight at 37°C under shaking. Cells were pelleted and resuspended in 1  Tris-HCl pH 8·0 in a volume equal to the cell pellet. A volume of 500 ll of the resulting suspension was transferred to a 1·5 ml screwcap polypropylene eppendorf tube. Subsequently, 500 ll phenol and 500 ll of 0·5 mm zirconium beads were added. The tubes were milled in a mini-bead beater for 2 min at high intensity. After milling, the tubes were stored on ice for at least 5 min and then centrifuged in an eppendorf centrifuge for 5 min at 10 000 rpm after which the aqueous phase was extracted with phenol-chloroform-isoamyl alcohol (ratio 25:24:1, respectively). After centrifugation the aqueous phase was transferred to a 2 ml eppendorf tube and was mixed with 2·5 volumes ice cold 96% ethanol and stored on ice. DNA was pelleted by centrifugation, washed with 70% ethanol, dried under vacuum and dissolved in 100 ll 10 m Tris-HCl pH 8·0, 1 m EDTA containing 100 lg ml−1 Ribonuclease A. This solution was incubated for 30 min at 37°C. A DNA concentration appropriate for the PCR is obtained by diluting this solution to a final concentration of 2 ng ll−1.

Polymerase chain reaction and restriction digest The DNA extracted from each Candida isolate was subjected to PCR amplification. The PCR was designed to amplify internal transcribed spacer (ITS) regions of the rDNA with established primers ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′) and NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′)9 in a reaction volume of 100 ll containing 2 m of each deoxynucleoside triphosphate, buffer mix containing (0·5  KCl, 1·5 m MgCl2, 0·2  Tris-HCl pH 7·7, 0·1% gelatin), 50 p of each primer, 5 U of AmplitaQ Polymerase (PE—Applied biosystems, North Warrington Cheshire, UK) and 2 ng of Candidal DNA. Negative controls were run with sterile deionized water. Reactions mixtures were subjected to 35 cycles of: denaturation at 94°C for 6 min, primer annealing at 55°C for 40 s, extention at 72°C for 2 min and final elongation step at 72°C for 5 min. An automated thermal cycler (Perkin Elmer 9600) was used in all experiments. The size and purity of the PCR products were tested by electrophoresis in a 0·7% agarose gel. DNA was visualized by staining with ethidium bromide and illumination with u.v. light. Polymerase chain reaction products were purified with 3  sodium acetate and 100% ethanol and were subsequently incubated at −80°C for 60 min. These were digested with 5 U of the restriction enzymes Bfal, Ddel or

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Fig. 1. PCR products with primers ITS 5 and NL4 of reference Candida species and patient isolates. Candida inconspicua from patients (lane 1) and (lane 2), C. inconspicua from reference strain number SBS180 to SBS4258 (lane 3 to lane 7), C. albicans reference number J930505 (lane 8), C. dubliniensis reference number 88/029 (lane 9), C. glabrata reference number 930562/2 (lane 10), C. guilliermondii reference numberJ932047/2 (lane 11), C. tropicalis reference number NCCLS 1 (lane 12), C. krusei reference number J930433 (lane 13), negative control (lane 14), molecular weight marker-100 bp DNA Ladder (lane M). Both the reference and patients strains gave exact the same PCR product.

HaeIII (New England Biolabs, Beverly, MA, USA) during overnight incubation at 37°C. The resulting restriction fragments were analysed by electrophoresis in a 2% agarose gel. The restriction profiles produced by these enzymes were analysed by the GELcompar package version 4·0.10 RESULTS Reference strains The internal transcribed spacer region was successfully amplified from all reference strains tested, and a distinct product size was consistently obtained for all isolates of a given species. All reference strains identified as C. glabrata, C. guilliermondii and C. inconspicua yielded unique product sizes of approximately 1500, 1300 and 1100 bp, respectively (Fig. 1). A product of approximately 1200 bp was obtained for the remaining Candida species, (C. albicans, C. dubliniensis, C. tropicalis and C. krusei), and their PCR products were treated with the restriction enzymes BfaI DdeI and HaeIII for further differentiation. Figure 2a–c shows a typical gel electrophoresis of the banding patterns obtained for seven Candida species after digestion of the PCR products.

could be differentiated from one another. All clinical isolates identified as C. glabrata, C. guilliermondii and C. inconspicua yielded unique product sizes of approximately 1500, 1300 and 1100 bp, respectively (Fig. 1). A product of approximately 1200 bp was obtained for the remaining Candida species, and their PCR products were treated with the restriction enzymes BfaI DdeI and HaeIII for further differentiation. The restriction profile of the ITS rDNA genes suggests that the difference (taking DdeI enzyme as an example) is due to a single DdeI site (band) change; that is, both have four DdeI fragments in the rDNA ITS repeat. In C. albicans, they are spaced at 450, 350, 210 and 150 bp, but in C. dubliniensis they are spaced at 450, 350, 210 and 110 bp (Fig. 2b, lane 8–9). Figure 2 a–c shows a typical gel electrophoresis of the banding patterns obtained for seven Candida species after digestion of the PCR products. In Table 1 we compare phenotypic and genotypic identifications of the 114 Candida isolates from 81 HIVinfected patients. Two out of 78 C. albicans, and one of 10 C. dubliniensis species was wrongly identified phenotypically. Table 1 shows the global results obtained for each isolate and repeated testing of a given isolate yielded reproducible and consistent result.

DISCUSSION Clinical isolates All isolates tested gave a consistent restriction fragment length polymorphism (RFLP) profile and species

The present study shows that seven medically important Candida species can be distinguished on the basis of size and structural differences in the rDNA

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Fig. 2. (a) (b) (c) Restriction digests of the PCR amplified ITS region of Candida species with (a) BfaI, (b) DdeI, (c) HaeIII. Patient sample C. inconspicua (lanes 1, 2), Reference samples C. inconspicua strain number SBS180 to SBS4258 (lanes 3–7), C. albicans reference number J930505 (lane 8), C. dubliniensis reference number 88/ 029 (lane 9), C. glabrata reference number 930562/2 (lane 10), C. guilliermondii reference number J932047/2 (lane 11), C. tropicalis reference number NCCLS 1 (lane 12), C. krusei reference number J930433 (lane 13), molecular weight marker-100 bp DNA Ladder (lane M). Both the reference and patients strains gave exact the same restriction fragment length polymorphism (RFLP) profiles except with C. inconspicua strains.

spacer regions. Isolates of C. glabrata, C. guilliermondii and C. inconspicua could be identified on the basis of PCR product size. Characteristic profiles, obtained following restriction digestion of PCR products, were necessary for the identification of the remaining species, C. albicans, C. krusei, C. tropicalis and C. dubliniensis (Fig. 2a–c) Both the reference and patients strains gave exact the same RFLP patterns except with C. inconspicua strains. In Fig. 2a and b, (lane 1–7) we detected some differences in the RFLP profiles of reference and patient C. inconspicua. The

third enzyme HaeIII Fig. 2c, (lane 1–7) did not detect any genetic differences between these strains. This might be due to polymorphisms or mutations that are present in the strains analysed, which changes the restriction site thereby resulting in a fragment of different molecular weight and size. The results obtained in the present study were reproducible and consistent for the isolates tested. The PCR applied here differs from those previously reported11–16 in that it is directed to the internal spacer (ITS) region between the small and large subunits of the rDNA gene. Williams et al.17 identified eight medically important Candida species by PCR-RFLP analysis. In addition to five of the seven species included in the present study, they identified three additional species: C. pseudotropicalis, C. parapsilosis and C. stellatoidea. Their method differ from ours in that they used the method described by Scherer and Stevens18 for the DNA extraction, whereas we developed a method for Candidal DNA extraction using the Bead-beater machine. Moreover, their PCR was designed to amplify the rDNA with established primers ITS19 which is at a different primer sequence position from ours. Results obtained with their PCR differed from ours: C. glabrata yielded a unique product size of 800 bp; all isolates of C. guillermondii and C. Pseudotropicalis yielded product sizes of 600 and 650 bp, respectively, and product of approximately 520 bp was obtained with the remaining isolates. We found the reference strains of Candida species examined by PCR-RFLP method, very homogenous interspecies and distinct within species. A striking finding was that C. dubliniensis and C. albicans are distinct by our method, although their biochemical phenotypes are very similar, differing from each other only by the b-glucosidase test. Candida dubliniensis may be underreported in clinical samples because most currently used isolation and identification methods fail to recognize this yeast.19 The restriction profile of the ITS rDNA genes suggests that the difference (taking DdeI enzyme as an example) is due to a single DdeI restriction site change in C. dubliniensis and C. albicans. One recent approach for establishing genetic relationships in medically important organisms is by RFLP analysis as reported by Magee et al.20 Several features of conventional restriction analyses limit their use in routine identification such as southern blot hybridization, requirement for relatively clean DNA as well as substantial time and resources for preparation of hybridization membranes and probes. Sensitivity of southern blots is sometimes affected by poor signal/noise ratio caused by poor hybridization of heterologuous probes and high background signals

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10 20 30 40 50 60 70 80 90 100 Bfa I

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bdh026 77 C. inconspicua

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bdh026 99 C. inconspicua

SBS4258

bdh026 44 C. inconspicua

SBS180

bdh026 55 C. inconspicua

SBS990

bdh026 66 C. inconspicua

SBS1735

bdh026 22 C. inconspicua

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bdh026 33 C. inconspicua

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bdh026 12 C. glabrata

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bdh026 15 C. krusei

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Fig. 3. Comparative analysis of electrophoresis patterns generated from RFLP profiles comprising the reference and patient Candida species. Patients samples are C. albicans 2, C. dubliniensis 3a, C. guilliermondii 74b, C. tropicalis 5b, Reference samples C. inconspicua strain number SBS2833 to SBS1735, Patient samples C. inconspicua 3c and 41, C. glabrata 5a, C. krusei 8c, molecular weight marker-100 bp DNA Ladder (lane M).

Table 1. Comparison between phenotypic and genotypic identifications of the 114 Candida isolates from 81 HIV-infected patients Patient isolates (n=114)

Candida species

C. C. C. C. C. C. C.

albicans dubliniensis glabrata tropicalis krusei inconspicua guilliermondii

Phenotypic identification

Genotypic identification

78 10 10 6 4 3 1

76 9 10 6 4 3 1

on blotting membranes. To circumvent these problems, we used an approach allowing direct analysis of the target sequence without the necessity for blotting or hybridization. We applied PCR to minute amounts of genomic DNA. If this is so, a simplified DNA preparation may be all that is required to place the technique at the disposal of clinical laboratory. In summary, we optimized a molecular method for the identification of seven Candida species using PCR to amplify a rDNA segment, and RFLP to further differentiate among the species. Sixty-five reference

Incorrect Actual species phenotypic (by genotypic identification analysis) 2 1

C. dubliniensis C. albicans

strains and 114 clinical isolates have been successfully analysed with three different frequent cutter restriction enzymes in this identification technique. The method is rapid and warrants further evaluation to other Candida species and fungi.

ACKNOWLEDGEMENTS The authors would like to thank their colleagues in the medical microbiology laboratory UIA: Christine Lammens,

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Patrick Descheemaeker, Marleen Van Looveren, Katherine Loens, Monik Wijdooghe, Machteld Hauchecorne, Peter Vandamme and Greet leven A note of appreciation to F.C. Odds (Mycology department, Janssen Research Foundation, Beerse) for providing us with the reference strains.

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11. Burgener-Kairuz, P., Zuber, J. P. & Jaunin, P. (1994). Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimen by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-alpha-demethylase (L1A1) gene fragment. Journal of Clinical Microbiology 32, 1902–7. 12. Fujita, S., Lasker, B. A. Lott, T. J. Reiss, E. & Morrison, C. J. (1995). Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood. Journal of Clinical Microbiology 33, 962–7. 13. Holmes, A., Cannon, R. D., Shepherd, M. G. & Jenkinson, H. F. (1994). Detection of Candida albicans and other yeasts in blood by PCR. Journal of Clinical Microbiology 32, 228–31. 14. Molina, F., Jong, S. C. & Huffman, J. L. (1993). PCR amplification of the 3′ external transcribed and intergenic spacer of the ribosomal DNA repeat unit in three species of Saccharomyces. FEMS Microbiology Letters 108, 259–64. 15. Ralph, D., McClelland, M., Welsh, J., Baranton, G. & Perolat, P. (1993). Leptospira species categorized by arbitrarily primed Polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCRamplified rRNA genes. Journal of Bacteriology 175, 973–81. 16. White, T., Arnheim, N. & Erlich, H. A. (1989). The Polymerase chain reaction. Trends Genetics 5, 185–9. 17. Williams, D., Wilson, M. J., Lewis, M. A. O. & Potts, J. C. (1995). Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer region of ribosomal DNA. Journal of Clinical Microbiology 33, 2476–9. 18. Scherer, S. & Stevens, D. A. (1987). Application of DNA typing methods to epidemiology and taxonomy of Candida species. Journal of Clinical Microbiology 25, 675–9. 19. Schoofs, A., Odds, F. C., Colebunders, R., leven, M. & Goossens, H. (1997). Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. European Journal of Clinical Microbiology and Infectious Disease 16, 296–300. 20. Magee, B., D’Souza, T. & Magee, M. P. T. (1987). Strain and species identification by restriction fragment length polymorphisms in the ribosomal DNA repeat of Candida species. Journal of Bacteriology 169, 1639–43.