Journal of Medical Microbiology Papers in Press. Published December 5, 2014 as doi:10.1099/jmm.0.078832-0

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Identification and characterization of nine atypical

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Candida dubliniensis clinical isolates

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Olatz Albaina 1, Ismail H. Sahand 1,5, María I. Brusca 2, Derek J. Sullivan 3,

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Iñigo Fernández de Larrinoa 4 and María D. Moragues 1,6

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1

Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y

Odontología, University of the Basque Country UPV/EHU, Bilbao (Spain). 2

Departamento de Microbiología y Parasitología, Facultad de Odontología, Universidad de

Buenos Aires (Argentina). 3

Microbiology Research Laboratory, Division of Oral Biosciences, School of Dental Science

and Dublin Dental University Hospital, Trinity College, Dublin 2 (Ireland). 4

Departamento de Química Aplicada, Facultad de Ciencias Químicas, University of the Basque

Country UPV/EHU, Donostia-San Sebastián (Spain). 5

Department of Microbiology, Faculty of Medicine, Hawler Medical University, Hawler,

Kurdistan (Iraq). 6

Departamento de Enfermería I, Escuela Universitaria de Enfermería, University of the Basque

Country UPV/EHU, Bilbao (Spain).

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*Corresponding author. Mailing address: Departamento de Enfermería I. Escuela Universitaria

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de Enfermería. University of the Basque Country, Barrio Sarriena s/n, 48940 Leioa, Vizcaya,

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Spain. E-mail: [email protected]

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Running title: Atypical Candida dubliniensis clinical isolates

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Abstract

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Candida dubliniensis is a pathogenic yeast of the genus Candida closely related to Candida

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albicans. The phenotypic similarity of these two species often leads to misidentification of C.

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dubliniensis isolates in clinical samples. DNA-based methods continue to be the most effective

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means of discriminating accurately between the two species. Here we report on the

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identification of nine unusual Candida isolates that showed ambiguous identification patterns on

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the basis of their phenotypic and immunological traits. The isolates were categorized into two

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groups. Group I isolates were unable to produce germ tubes and chlamydospores and to

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agglutinate commercial latex particles coated with a monoclonal antibody highly specific for C.

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dubliniensis. Group II isolates grew as pink and white colonies on CHROMagarTM Candida and

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ChromIDTM Candida respectively. Carbohydrate assimilation profiles obtained with API ID

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32C® together with PCR amplification with specific primers and DNA sequencing allowed a

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reliable identification of the nine unusual clinical isolates as Candida dubliniensis.

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Keywords: Candida dubliniensis, uncommon isolates, phenotypic identification, genotypic

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identification, immunological identification.

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Introduction

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The incidence of systemic and mucosal infections caused by species of the genus Candida has

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increased dramatically in recent decades. Candida albicans is considered the most pathogenic

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species of the genus Candida and the most common cause of deep and superficial candidiasis

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(Coleman et al., 1997b). Besides the elevated prevalence of infections caused by C. albicans,

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other species such as Candida glabrata, Candida tropicalis, Candida dubliniensis, Candida

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parapsilosis, Candida krusei and Candida guilliermondii are also associated with invasive

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candidiasis in humans (Krcmery & Barnes, 2002) .

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C. dubliniensis is an opportunistic yeast of the genus Candida primarily associated with oral

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candidiasis (Sullivan & Coleman, 1998, Sullivan et al., 1995) in human patients infected with

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immunodeficiency virus (HIV), or with AIDS (Gugnani et al., 2003, Patel et al., 2012, Sullivan

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et al., 2004, Sullivan et al., 2004). In addition, this species has been described to a lesser extent

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in healthy individuals and people non-infected with HIV but affected by severe underlying

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illnesses such as diabetes, cystic fibrosis or cancer (Coleman et al., 1997b, Dahiya et al., 2003,

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Davies et al., 2002, Lai et al., 2013, Manfredi et al., 2002, Parmeland et al., 2013, Peltroche-

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Llacsahuanga et al., 2002, Willis et al., 2000, Yu et al., 2012).

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Genome sequence analysis has confirmed the very close relationship between C. dubliniensis

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and C. albicans (Jackson et al., 2009) and, unsurprisingly, the two species share many

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phenotypic traits which complicate the identification of C. dubliniensis clinical isolates by

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conventional mycological methods. In order to distinguish between these two Candida species

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several identification assays, with varying degrees of accuracy, based on phenotypic, genotypic

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and immunological differences are available. Phenotypic tests such as the ability to grow at

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different temperatures, colony color and morphology on chromogenic media, chlamydospore

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production or carbohydrate assimilation tests are widely used as routine assays in clinical

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microbiology laboratories. In addition, a commercial test (Bichro-Dubli Fumouze®) to identify

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C. dubliniensis isolates by latex-agglutination using beads sensitized with a highly specific

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monoclonal antibody (mAb 12F7-F2) (Marot-Leblond et al., 2006) is currently available.

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However, the most accurate and reliable tests to discriminate between C. dubliniensis and C.

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albicans are those based on DNA analysis, such as DNA amplification by PCR with specific

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primers and DNA sequencing applied to the ribosomal RNA gene cluster (Donnelly et al., 1999,

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Ellepola et al., 2003).

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Here we report on a population study with 477 clinical isolates of Candida spp. where 434 of

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them were identified as C. albicans, 34 isolates were C. dubliniensis and nine isolates showed

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anomalous patterns on various routine identification assays. DNA analysis allowed the

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unambiguous identification of all these uncommon isolates as Candida dubliniensis, whose

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phenotypic and genotypic properties are discussed in this work.

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Materials and Methods

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Isolates and culture conditions. A total of 477 clinical isolates of Candida spp. recovered from

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different anatomical sites (oral mucosa, blood, respiratory tract) of patients from disparate

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geographic locations were included in this study. Among these were 434 C. albicans and 34 C.

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dubliniensis isolates as well as nine atypical isolates which we were unable to identify using

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routine diagnostic tests due to their unusual phenotype.

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The geographical and clinical origins of the nine unusual isolates identified amongst this strain

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collection are listed in Table 1. Group I isolates (n = 3) were collected from the oral cavity of

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three separate HIV-infected patients in Spain and group II isolates (n = 6) were collected from

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the oral cavity of six different individuals with gingival or periodontal disorders in Argentina.

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Reference strains for identification tests included C. dubliniensis NCPF 3949 obtained from the

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National Collection of Pathogenic Fungi (NCPF, Bristol, UK) and C. albicans SC5314 obtained

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from the Stanford DNA Sequencing and Technology Center (SC, Stanford, USA). Reference

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strains belonging to the C. dubliniensis genotypes 1, 2, 3 and 4 were respectively NCPF 3949,

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CBS 2747 obtained from the Centraalbureau Voor Schimmelcultures (CBS, Utrecht, The

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Netherlands), and clinical isolates p6265 and p7718 (Gee et al., 2002) obtained from Dublin

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Dental University Hospital. Yeasts were grown routinely on Sabouraud dextrose agar (SDA;

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Difco, Detroit, MI, USA) for 24-48 h at 37 ºC.

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Phenotypic analyses. Germ tube tests were performed in fetal bovine serum (FBS; Sigma, St.

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Louis, MO, USA) either concentrated or diluted with varying amounts of 0.65% (w/v) yeast

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nitrogen base minimal medium without amino acids (YNB; Difco, Detroit, MI, USA)

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containing 5% (w/v) glucose. One million yeast cells were inoculated in 0.5 ml of pure or

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diluted FBS, incubated at 37 ºC for 3 h, and germ tube formation was observed by microscopy.

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The ability of isolates to grow at elevated temperature was determined by examining their

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ability to grow on SDA plates incubated at 45 ºC for 24-48 h.

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Chlamydospore production was examined by incubating the isolates for 72 h at 30 ºC on Pal‘s

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agar (Pal, 1997), Staib agar (Staib & Morschhauser, 1999), Casein agar (Mosca et al., 2003),

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Cornmeal agar (Sullivan et al., 1995), CHROM-Pal´s agar (CH-P) (Sahand et al., 2005), and

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YNB agar supplemented with galactose 0.025% (w/v) and methionine (25 mg l-1 to 100 mg l-1)

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(Citiulo et al., 2009).

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Isolates were also cultured on a range of chromogenic media such as CHROMagarTM Candida

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(CAC; CHROMagar Company, Paris, France), CH-P and ChromIDTM Candida chromogenic

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media (ChromID; bioMérieux, Marcy l’Etoile, France) at 30 ºC (CH-P) or 37 °C (CAC;

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ChromID) for 48 h.

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Phenotypic variability of the colonies was also examined on SDA supplemented with phloxine

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B (5 mg l-1 Sigma Chemical Co. USA; (Lipperheide et al., 2002). Plates were inoculated with

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approximately 100 cells and incubated for up to ten days at 37 °C.

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Carbohydrate assimilation profiles were determined by using the API ID 32C® yeast

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identification system (bioMérieux, Marcy l’Etoile, France), according to the manufacturer’s

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instructions.

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Immunological characterization

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The Bichro-Dubli Fumouze® latex agglutination test (Fumouze Diagnostics, Levallois-Perret,

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France) for identification of C. dubliniensis strains was performed according to the

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manufacturer’s instructions (Sahand et al., 2006). Indirect immunofluorescence assays (IFA)

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were performed as described by Bikandi et al. (Bikandi et al., 1998) using a purified preparation

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of mAb 12F7-F2 (1.49 mg ml-1) kindly provided by Dr. Raymond Robert (Angers, France),

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and a polyclonal anti-C. dubliniensis serum raised in a rabbit infected with C. dubliniensis

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which was adsorbed 1:1 with formalin-fixed C. albicans NCPF 3153 blastospores as described

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by the same authors (Bikandi et al., 1998). Briefly, blastospores were grown on SDA plates for

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48 h at 24 °C, resuspended in PBS at a cell density of 106 cells ml-1 and fixed on Teflon-coated

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immunofluorescence slides. These slides were incubated for 30 min at 37°C with 10 µl of mAb

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12F7-F2 diluted 1:50 in PBS or 1:5 in the case of the adsorbed anti-C. dubliniensis serum. After

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washing, a second incubation was perfomed with 10 µl of anti-mouse IgG FITC (Sigma, St.

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Louis, MO, USA) diluted 1:250 in PBS-TA [PBS supplemented with Evans blue (0.05 %, w/v)

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and Tween 20 (0.05 % v/v)] for mAb 12F7-F2, and in the case of anti-C. dubliniensis serum the

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second incubation was performed under the same conditions using 10 µl of anti-rabbit IgG

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FITC conjugated antibody (Sigma, St. Louis, MO, USA) diluted 1:250 in PBS-TA. Slides were

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mounted with bicarbonate buffered glycerol mounting fluid and examined with a Nikon Eclipse

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80i epifluorescence microscope.

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DNA extraction

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Yeast genomic DNA was extracted as described by Hoffman and Winston (Hoffman &

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Winston, 1987), with minor changes. Briefly, yeast cells were grown overnight in 5 ml of

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Sabouraud broth at 37 °C and 120 r.p.m. in a Gallenkamp orbital shaker incubator (Gemini,

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Apeldoorn, Netherlands). 1.5 ml of culture was harvested by centrifugation at 14000 r.p.m.

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(Heraeus Biofuge Pico microcentrifuge, DJBLabcare, Buckinghamshire, UK) and the pellet was

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washed twice with sterile MilliQ water. Cells were lysed in 200 μl breaking buffer (2% (v/v)

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Triton X-100, 1% (w/v) SDS, 100 mM NaCl, 10 mM Tris-Cl, pH 8.0 and 1 mM EDTA pH 8.0)

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by vortexing with 0.3 g of glass beads (0.5 mm diameter, Sigma, St. Louis, MO, USA) five

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times for 1 min separated with identical intervals of cooling on ice. Five hundred microliters of

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a phenol: chloroform: isoamyl alcohol (25:24:1, Sigma, St. Louis, MO, USA) solution was

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added to the lysate and the mixture was homogenized vigorously for 30 seconds. At this point,

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samples were centrifuged at 14000 r.p.m. for 10 min (Heraeus Biofuge Pico microcentrifuge)

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and the upper layer was transferred to a new vial containing 200 µl of the phenol: chloroform:

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isoamyl alcohol solution. The mixture was homogenized and centrifuged again at 14000 r.p.m.

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for 3 min. The upper phase was collected and after the addition of 20 µl of 3 M sodium acetate

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and 400 µl of cold ethanol (-20 °C), the vials were inverted several times until precipitated

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DNA was visualized. Subsequently, the samples were centrifuged for 5 min at 14000 r.p.m. and

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the supernatants decanted. Pellets were resuspended in 1 ml of cold 70% ethanol and

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centrifuged for 1 min at 14000 r.p.m. Supernatants were decanted again and DNA pellets were

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resuspended in 50 µl of TE buffer (10 mMTris-Cl, pH 8.0 and 1 mM EDTA pH 8.0).

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Genotypic characterization

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Further characterization of test isolates was conducted using multiplex PCR amplification with

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two pairs of specific primers. One pair of primers, CALB1F and CALB2R, specific for C.

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albicans, amplified a fragment of 273 bp from the internal transcribed spacer regions ITS1 and

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ITS2 of the rRNA genes (Luo & Mitchell, 2002), while the second one, CDBF28F and

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CDBR110R highly specific for C. dubliniensis, amplified an 816 bp fragment of the

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topoisomerase II gene (Kanbe et al., 2003). In addition, a conventional PCR assay was

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performed to discriminate among Candida africana, C. albicans and C. dubliniensis by means

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of a set of primers (CR-f and CR-r) derived from gene HWP1 as described by Romeo and

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Criseo (Romeo & Criseo, 2008).

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The identity of the isolates tested was assigned by sequencing the large subunit ribosomal DNA

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D1/D2 domain after amplification with NL1 and NL4 primers, as described by Kurtzman and

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Robnett (Kurtzman & Robnett, 1998). Sequence alignment was performed using the BLAST

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tool at the National Center for Biotechnology Information (NCBI, Bethesda, MD). The

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assignment of genotype for the C. dubliniensis isolates was performed by PCR with primers

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G1F/G1R, G2F/G2R, G3F/G3R and G4F/G4R according to the method of Gee and colleagues

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(Gee et al., 2002) as described by Brena et al. (Brena et al., 2004), and the four reference strains

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of C. dubliniensis genotypes 1, 2, 3 and 4 (NCPF 3949, CBS 2747, p6265 and p7718

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respectively) were used as positive controls.

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In order to determine the genotype of the uncommon C. dubliniensis isolates at the MTL

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(Mating type like) locus, a multiplex PCR using two pairs of specific primers was performed as

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described by Rustad et al. (Rustad et al., 2002). One pair of primers, MTLa1-F and MTLa1-R,

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amplified a fragment of 535 bp and the second one, MTLα1-F and MTLα1-R, rendered a

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product of 615 bp.

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Antifungal susceptibility testing

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The susceptibility of isolates to current antifungal drugs was tested by using the commercial

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Sensititre Yeast One 8® (TREK Diagnostic Systems, Cleveland, OH, USA) colorimetric method

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according to the manufacturer’s instructions. Plates contained serial dilutions of eight

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dehydrated compounds: amphotericin B (0.008 to 16 mg l-1), fluconazole (0.125-256 mg l-1),

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itraconazole (0.008-16 mg l-1), ketoconazole (0.008-16 mg l-1), voriconazole (0.008-16 mg l-1),

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posaconazole (0.008-8 mg l-1), 5-fluorocytosine (0.03-64 mg l-1) and caspofungin (0.008-16 mg

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l-1). Each well incorporated the fluorescent dye Alamar Blue (resazurin) which turned from blue

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to pink when microbial growth occurred.

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Results

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A total of 477 clinical isolates first identified as C. albicans that had been recovered from

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different anatomical sites (oral mucosa, blood, respiratory tract) of patients from disparate

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geographic locations, were revised in order to investigate the prevalence of C. dubliniensis

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among them. By so doing, 34 strains mostly isolated from oral samples were renamed as C.

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dubliniensis. During this process we also found nine additional oral isolates with unusual

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phenotypic traits which hampered their definitive identification (Table 1). Further

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characterization of these nine isolates was performed with several phenotypic, genotypic, and

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immunological assays and the results obtained were matched against reference strains of C.

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dubliniensis and C. albicans.

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Phenotypic characteristics. A summary of the results obtained in the phenotypic analysis of

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the nine isolates is shown in Table 2. While all nine isolates grew well as smooth creamy white

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colonies at 30 ºC and 37 °C on SDA medium, they did not show signs of growth when

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incubated at 45 °C. When examined microscopically, six isolates (08-15 to 08-20) formed round

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and ovoid shaped blastospores, similar to those of the reference strains C. dubliniensis NCPF

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3949 and C. albicans SC5314, whereas yeast cells from the remaining three isolates (98-277,

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95-677 and 94-234) were smaller with ovoid, narrow and elongated shapes (Figure 1). These

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differences in blastospore morphology were used as a trait to categorize the uncommon isolates

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into two groups: Group I included the three isolates 98-277, 95-677 and 94-234, while the

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remaining six were allocated to group II.

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Group II isolates were found to produce germ tubes in 10% FBS, and abundant chlamydospores

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on Staib agar, Pal agar, Corn meal agar or Casein agar. In contrast, group I isolates were unable

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to form germ tubes even when exposed to 100% FBS. In addition, the latter isolates did not

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form chlamydospores in the above mentioned media (Figure 2) nor even in YNB medium

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supplemented with galactose and methionine (25 mg l-1) which has been formulated as a

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suitable medium for inducing abundant chlamydospores formation in C. dubliniensis but not in

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C. albicans. Moreover, increasing methionine concentration up to 100 mg l-1 did not modify the

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phenotype shown by group I isolates.

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The results obtained by culturing the test isolates on chromogenic media are illustrated in Figure

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3. The colony colors of isolates grown on CAC medium were compared to the dark green color

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characteristic of C. dubliniensis and to the light green color of C. albicans, which were used as

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controls. Group I isolates developed different tones of green, while the remaining unusual

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strains formed yellowish-white or pink colonies on this medium. Assays were also performed

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on CH-P medium plates where C. dubliniensis isolates produce rough bluish-green colonies,

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whereas most of C. albicans isolates usually grow as smooth light-green colonies (Sahand et al.,

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2005, Sahand et al., 2009). On this medium group I isolates yielded different shades of bluish-

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green and light-green smooth colonies, whereas group II isolates developed pink rough

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colonies.

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On ChromID agar most of C. albicans colonies appear cobalt blue, whilst C. dubliniensis

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isolates form turquoise colonies. However, while group I isolates developed turquoise and

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bluish-white colonies, the remaining six unusual isolates grew as white colonies.

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On SDA agar supplemented with phloxine B the unusual isolates and reference strains displayed

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distinct colonial phenotypic variants (Figure 3). All the colonies examined reached a similar

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size with varied colors and texture, except isolate 94-234 which exhibited the smallest colonies.

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Group II isolates showed a rather homogeneous phenotype with mostly smooth pink-coloured

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colonies. In contrast, the colonies of group I isolates were more diverse, with smooth, rough or

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wrinkled colonies (strain 95-677), colors from pale to dark pink, and sometimes differential

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sectors in the same colony (strain 98-277) reminiscent of the white-opaque transition.

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The following API ID 32C® codes were obtained for the nine unusual isolates: 7142140015,

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7142100215, 7143140015 and 7342140015. All of these codes have been described previously

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for isolates of C. dubliniensis. As with most C. dubliniensis strains, our test isolates were unable

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to assimilate methyl-α-D-glucoside, lactate, trehalose or xylose, with the exceptions of the 94-

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234 isolate which was able to use trehalose as carbon source, and the six uncommon isolates of

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group II which were able to grow on lactate. However, these data strongly suggest that the nine

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unusual isolates are C. dubliniensis.

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Immunological characteristics. Bichro-Dubli Fumouze® (Marot-Leblond et al., 2006) is a

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commercial latex agglutination assay based on the specificity of the mAb 12F7-F2 to bind to C.

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dubliniensis cells and not to other Candida species, thus allowing an easy identification of C.

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dubliniensis among clinical isolates. While the group II isolates yielded a positive reaction using

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this test, the three group I isolates were negative. The reactivity of the mAb 12F7-F2 with the

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uncommon isolates was further examined by indirect immunofluorescence microscopy using a

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purified preparation of this antibody. As expected, group II isolates emitted intense fluorescence

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under the microscope whereas group I isolates were not labeled by the mAb 12F7-F2. However,

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all group I isolates emitted strong fluorescence after reacting with a C. albicans-adsorbed

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polyclonal antiserum raised in a rabbit experimentally infected with C. dubliniensis NCPF 3949

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(Bikandi et al., 1998), while the reactivity of group II isolates was heterogeneous (Table 2).

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Genotypic characteristics. Results from genotypic analysis assays are summarized in Table 2.

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PCR-amplification of the HWP1 gene yielded a 569 bp DNA fragment for all the test samples

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as well as for the C. dubliniensis reference strain (Figure 4), while the reference C. albicans

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amplicon size was 941 bp. The identity of the atypical strains as C. dubliniensis was confirmed

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by multiplex PCR on the basis of differences in their ITS region and topoisomerase II gene, that

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allowed for the identification of C. albicans and C. dubliniensis isolates simultaneously. The

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amplified DNA fragments of all the unusual isolates were similar in size to the 816 bp amplicon

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obtained for the C. dubliniensis reference strain. By sequencing the D1/D2 domain of the large

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subunit ribosomal DNA we obtained three sequences corresponding to the group I isolates,

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which are now available at the NCBI sequence repository under accession numbers KF537273,

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KF537274 and KF537275. As a result of comparative sequence analyses, the unusual isolates

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displayed 99% homology to C. dubliniensis sequences stored in NCBI database. The analysis of

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genotypes by the method of Gee et al. (Gee et al., 2002) revealed that group I isolates belonged

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to genotype 1, while the remaining atypical isolates belonged to genotype 2.

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Finally, all the uncommon clinical isolates of C. dubliniensis were characterized as

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heterozygous MTLaα.

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Susceptibility to antifungal drugs. Table 3 shows the minimum inhibitory concentration

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(MIC) values corresponding to the uncommon isolates under study for the eight antifungal

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drugs offered by the Sensititre YeastOne 8® system.

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According to the Clinical and Laboratory Standards Institute (CLSI) guidelines and

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recommended cut off values, all the uncommon isolates were categorized as “susceptible” to the

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set of antifungal drugs tested. Interestingly, when compared to the reference strain C.

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dubliniensis NCPF 3949 the uncommon isolates displayed even increased susceptibility to

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fluconazole and amphotericin B; in particular, group I isolates were the most susceptible to

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amphotericin B (MIC range 0.06-0.128 μg ml-1), and those of group II to fluconazole (0.25 μg

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ml-1) together with strain 95-677.

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Discussion

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The identification of Candida species with a simple, specific, and cost-effective assay is

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essential in medical mycology laboratories in order to achieve a rapid diagnosis and to initiate a

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suitable antifungal therapy as soon as possible. In a population of 477 clinical isolates which

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were grown on CHROMagarTM Candida, 434 isolates were identified as C. albicans, 34 as C.

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dubliniensis, and nine isolates yielded anomalous results when subjected to phenotypic analysis.

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While some phenotypic tests generated ambiguous results, “gold-standard” DNA analysis by

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PCR-amplification methods based on differences in the sequences of HWP1 and topoisomerase

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II genes and sequence analysis of the D1/D2 domain of the large subunit ribosomal RNA gene

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(rDNA) permitted the definitive identification of the nine isolates as Candida dubliniensis.

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Several authors have previously reported the specificity and suitability of these methods to

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identify clinical isolates of C. dubliniensis (Bosco-Borgeat et al., 2011, Gasparoto et al., 2009,

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Khlif et al., 2009, Marcos-Arias et al., 2009, Sahand et al., 2009).

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Clearly some phenotypic tests accurately identified the unusual isolates correctly. Unlike many

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C. albicans isolates, C. dubliniensis cannot grow on agar media at 45 °C, and this condition is a

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simple phenotype commonly used to discriminate these two Candida species. The inability of

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all the uncommon isolates to grow at 45 °C suggests that they fit the pattern established for C.

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dubliniensis. However, this phenotype is not considered conclusive because C. albicans isolates

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that are unable to grow, or grow poorly, at 45 °C have been reported as well (Gales et al., 1999,

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Kurzai et al., 2000, Pinjon et al., 1998, Schoofs et al., 1997, Sullivan & Coleman, 1998).

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Several complex culture conditions have been developed where C. dubliniensis can induce

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chlamydospore formation with high efficiency while C. albicans cannot (Mosca et al., 2003,

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Pal, 1997, Staib & Morschhauser, 1999, Sullivan et al., 1995). In such media, group II isolates

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and the reference C. dubliniensis strain produced abundant chlamydospores whereas,

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unexpectedly, group I isolates failed to form these thick-walled spherical structures. Even in a

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synthetic medium (Citiulo et al., 2009) that provides optimal conditions to stimulate abundant

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chlamydospore formation in C. dubliniensis, the group I isolates still failed to generate

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chlamydospores.

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In addition, the unusual isolates were capable to switch their colony morphology similarly to the

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white-opaque switching observed for the reference strains of C. dubliniensis and C. albicans.

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Even more group I isolates seemed to adopt spontaneously aberrant colony morphologies, most

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likely as a result of a different morphogenetic event based on a second switching system. The

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suggestive idea that group I isolates are morphogenesis-defective cells harboring deficiencies in

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one or more morphogenetic regulatory pathways (Staib & Morschhauser, 2007) cannot be

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discarded.

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Several chromogenic media are now available to differentiate C. albicans from C. dubliniensis.

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Many authors have verified the suitability of CAC medium to differentiate these two Candida

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species on the basis of the green color produced by their respective colonies (Baumgartner et

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al., 1996, Bernal et al., 1996, Casal et al., 1997, Coleman et al., 1997a, Coleman et al., 1997b,

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Garcia-Martos et al., 1998, Giusiano & Mangiaterra, 1998, Willinger et al., 2001). In our hands,

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group I isolates showed different shades of green color that hindered their positive identification

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as C. albicans or C. dubliniensis. Our results for this group of strains are consistent with other

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reports in the literature (Eraso et al., 2006a, Pincus et al., 1999, Tintelnot et al., 2000). The

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remaining uncommon isolates (group II) developed pink colonies which is inconsistent with

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either C. albicans or C. dubliniensis. To our knowledge, only Odds and Davidson (Odds &

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Davidson, 2000) had previously described some C. albicans isolates that formed pink colored

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colonies on CAC medium. It has been reported that differentiation between C. dubliniensis and

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C. albicans clinical isolates can be improved in CH-P agar (Sahand et al., 2005, Sahand et al.,

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2009), however in our hands both media, CH-P and CAC, lacked discriminatory capacity to

338

distinguish both Candida species accurately, when dealing with these unusual isolates. Finally,

339

ChromID Candida medium has high sensitivity and specificity to differentiate C. dubliniensis

340

from C. albicans (Eraso et al., 2006b). In this medium group I isolates resembled the C.

341

dubliniensis reference strain, whilst group II isolates grew anomalously as white colonies.

15

342

Interestingly, Eraso et al. (Eraso et al., 2006a) have previously described C. dubliniensis isolates

343

showing white colored colonies on this medium.

344

The single phenotypic test which enabled a clear differentiation of the nine uncommon isolates

345

and could be useful for strain identification was the commercial API ID 32C® system on the

346

basis of differences in carbohydrate assimilation profiles. All the numerical profiles recorded for

347

the uncommon isolates had already been described for C. dubliniensis strains (Cardenes-Perera

348

et al., 2004, Moran et al., 1997).

349

Based on phenotypic tests it is clear that group II isolates should be classified as C. dubliniensis,

350

however, the identity of the group I isolates could be considered inconclusive and suggests that,

351

with the exception of API ID 32C, phenotypic tests may be illustrative but not conclusive for

352

the differentiation between C. dubliniensis and C. albicans (Gales et al., 1999, Silveira-Gomes

353

et al., 2011).

354

The immunoagglutination test Bichro-Dubli Fumouze® indicated that group II isolates should be

355

classified as C. dubliniensis but group I isolates should not (Sahand et al., 2006). This

356

classification was in accordance with the immunofluorescence microscopy assay developed

357

with a purified preparation of 12F7-F2 mAb. However, all the isolates categorized as group I

358

showed intense reactivity with a rabbit polyclonal anti-C. dubliniensis specific antiserum,

359

confirming their identity as this species. This behavior may be due to its polyclonal nature,

360

recognizing antigens different to that specific for mAb 12F7-F2. Furthermore, regarding the

361

extremely weak reaction of group I isolates with the mAb 12F7-F2 and their inability to form

362

chlamydospores and germ tubes, a hypothetical relation between the specific antigen recognized

363

by the mAb (Marot-Leblond et al., 2006) and the switching process of C. dubliniensis cannot be

364

discarded. Since the cells of group I isolates grown in liquid culture are not typical ovoid yeast-

365

shaped, it is possible that these cells represent a switched form, perhaps similar to C. albicans

366

opaque cells. If so it may be possible that the antigen recognized by mAb 12F7-F2 is only

367

present on normal ovoid yeast cells and absent from the surface of switched derivatives, such as

368

the group I isolates. Altogether, the results of immunological assays are not conclusive and the

16

369

most reliable approach to identify these atypical isolates was obtained with molecular methods

370

of DNA analysis. These methods also enabled further characterization of the nine uncommon

371

isolates, providing relevant data for C. dubliniensis epidemiology. Thus, group I isolates

372

belonged to genotype 1, which is the most common amongst C. dubliniensis isolates (Gee et al.,

373

2002), while group II isolates belonged to genotype 2 which ranked second in frequency (Brena

374

et al., 2004, Gee et al., 2002). In addition, the nine strains were classified as heterozygous

375

MTLaα, which is the most common mating type among C. dubliniensis and C. albicans strains

376

(Pujol et al., 2004).

377

In summary, the presence of atypical fungal isolates in clinical samples and their appearance as

378

false negative results in various identification assays used for the routine analysis in the clinical

379

mycology laboratory, reinforce the necessity of performing molecular methods of DNA analysis

380

to ascertain the identity of fungal isolates, including the uncommon ones. Fortunately, while

381

these atypical isolates may represent a diagnostic challenge, since they are all susceptible to the

382

most commonly used antifungal agents they do not represent a therapeutic challenge as well.

383

17

384 385

Acknowledgements

386

This work was supported by grants IT-264-07 and IT-788-13 from Department of Education,

387

Universities and Research, and S-PE08UN10 Department of Industry, Innovation, Trade and

388

Tourism of the Basque Government, and UFI11/25 of the University of the Basque Country

389

UPV/EHU. Olatz Albaina was supported by a grant from the University of the Basque Country

390

UPV/EHU (Ayudas para la Formación de Personal Investigador, PIFG/005/07). Authors are

391

very grateful to Dr. Raymond Robert for supplying the monoclonal antibody 12F7-F2 and to

392

Cristina Marcos-Arias, Inés Arrieta-Aguirre and Giula Carrano for their technical assistance

393

with photography. Technical and human support provided by SGIker (UPV/EHU, MICINN,

394

GV/EJ, ERDF and ESF), is gratefully acknowledged.

395

This work is dedicated to the memory of Prof. José Pontón, unforgettable colleague and friend.

18

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24

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Fumouze

to

distinguish

Candida

dubliniensis

from

Candida

albicans.

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25

Willinger, B., Hillowoth, C., Selitsch, B. & Manafi, M. (2001). Performance of candida ID, a new chromogenic medium for presumptive identification of Candida species, in comparison to CHROMagar Candida. J.Clin.Microbiol. 39, 3793-3795. Willis, A. M., Coulter, W. A., Sullivan, D. J., Coleman, D. C., Hayes, J. R., Bell, P. M. & Lamey, P. J. (2000). Isolation of C. dubliniensis from insulin-using diabetes mellitus patients. J.Oral Pathol.Med. 29, 86-90. Yu, N., Kim, H. R. & Lee, M. K. (2012). The first Korean case of candidemia due to Candida dubliniensis. Ann.Lab.Med. 32, 225-228.

26

FIGURE LEGENDS Figure 1. Phase-contrast microscopy of the blastospores of reference strains (A) C. dubliniensis NCPF 3949 and (B) C. albicans SC5314, and group I isolates (C) 98-277, (D) 95-677 and (E) 94-234, grown on SDA at 37 ºC for 24 h. Figure 2. Cells of C. albicans and C. dubliniensis reference strains and atypical isolates grown on CHROMagar-Pal’s agar at 30 ºC for 72 h so as to develop chlamydospores (arrows). C. dubliniensis NCPF 3949, C. albicans SC5314, atypical group I isolates (98-277, 95-677 and 94-234) and group II isolates (08-15, 08-16, 08-17, 08-18, 08-19 and 08-20). Figure 3. Representative colonies of the unusual isolates and the reference strains C. dubliniensis NCPF 3949 and C. albicans SC5314 grown on various differential media and incubation conditions. CAC and ChromID media at 37 ºC for 48h, and CH-P medium at 30 ºC for 72h; SDA-phloxine B 9-10 days at 37 ºC. Figure 4. PCR amplification products obtained with primers CR-f/CR-r (HWP1 gene). Lanes 1-3, group I isolates: 98-277, 95-677 and 94-234; lanes 4-9, group II isolates: 08-15 to 08-20; lane 10: C. albicans SC5314; lane 11: C. dubliniensis NCPF 3949; lane M: Molecular size marker HyperLadder IV (Bioline, London, UK).

27

TABLES Table 1. Geographical origin of unusual Candida dubliniensis oral isolates and underlying clinical condition of patients

Group I

Group II

Underlying patient condition Oral infection HIV positive Oral infection HIV positive Oral infection HIV positive

Isolates

Country of origin

Year of isolation

94-234

Spain

1994

95-677

Spain

1995

98-277

Spain

1998

08-15

Argentina

2008

Gingivitis

08-16

Argentina

2008

Periodontitis

08-17

Argentina

2008

Periodontitis

08-18

Argentina

2008

Periodontitis

08-19

Argentina

2008

Periodontitis

08-20

Argentina

2008

Gingivitis

28

Table 2. Phenotypic, immunological and genotypic characteristics of the unusual isolates of C. dubliniensis in relation to the reference strains C. dubliniensis NCPF 3949 and C. albicans SC5314

Germ tube 45 ºC CAC CH-P ChromID

C. dubliniensis NCPF 3949

C. albicans SC5314

98-277

Group I 95-677

94-234

08-15

08-16

08-17

08-18

08-19

08-20

+ dark green bluish green turquoise

+ + green pale blue cobalt blue

dark green blue turquoise

green bluish green turquoise

light green light green bluish white

+ pink pink white

+ pink pink white

+ pink pink white

+ pink pink white

+ pink pink white

+ pink pink white

Group II

Chlamydospores + + + + + + + ID32C 7142100015 7347340015 7142100215 7142140015 7143140015 7342140015 7342140015 7342140015 7342140015 7342140015 7342140015 Bichro-Dubli + + + + + + + IFA 12F7-F2 + + + + + + + IFA anti-C.d. + + + + + + weak weak PCR HWP1 569 bp 941 bp 569 bp 569 bp 569 bp 569 bp 569 bp 569 bp 569 bp 569 bp 569 bp PCR TOP2 816 bp NA 816 bp 816 bp 816 bp 816 bp 816 bp 816 bp 816 bp 816 bp 816 bp Sequence C.d. C.a. C.d. C.d. C.d. C.d. C.d. C.d. C.d. C.d. C.d. C.d. Genotype 1 NA 1 1 1 2 2 2 2 2 2 MTL aα aα aα aα aα aα aα aα aα aα aα 1 C.d.: C. dubliniensis; C.a.: C. albicans ;(+): positive; (-): negative; NA, not applicable

29

1 2

3 4

Table 3. Susceptibility to antifungal drugs (MIC; µg ml-1) of the unusual isolates of C. dubliniensis in relation to the reference strain NCPF 3949 and C. albicans SC5314.

FCZ

C. dubliniensis NCPF 3949 1

ITZ

0.016

0.06

0.06

0.016

≤0.008

0.032

0.032

0.064

0.064

0.064

0.064

VCZ

0.016

0.015

≤0.008

0.016

0.03

≤0.008

≤0.008

≤0.008

≤0.008

0.016

≤0.008

PCZ

0.016

0.03

0.016

0.016

0.03

0.016

0.016

0.032

0.032

0.032

0.064

KTZ

0.016

n.a.

0.016

0.016

≤0.008

≤0.008

≤0.008

≤0.008

≤0.008

≤0.008

≤0.008

AMB

1

1

0.128

0.06

0.128

0.5

0.5

0.5

0.5

0.5

0.5

5-FC

≤0.03