Isolation of Candida dubliniensis in a teenager with denture stomatitis

MOSCA CO, MORAGUES MD, BRENA S, ROSA AC, PONTÓN J. ISOLATION OF CANDIDA DUBLINIENSIS IN A TEENAGER WITH DENTURE STOMATITIS. MED ORAL PATOL ORAL CIR BUCAL 2005;10:25-31.

SUMMARY

Objectives: Test several methods that allow the differentiation between Candida albicans and Candida dubliniensis, in an attempt to assess whether C. dubliniensis can be recovered from the oral cavity of teenagers wearing orthopedic oral prostheses. 
Material and Methods: Twelve Candida strains were isolated from the prosthesis as well as the palatal mucosa in contact with the dental prosthesis from 12 teenager patients wearing orthopedic oral prostheses. Differentiation between C. albicans and C. dubliniensis was achieved by a number of phenotypic tests (carbon assimilation by the commercially available ID 32C test, growth at 45ºC on Sabouraud glucose agar, abundant chlamydospore production on Casein agar, and reactivity with a C. dubliniensis antiserum) and the polymerase chain reaction (PCR). Serotyping of C. albicans was performed with monoclonal antibody B9E.
Results: All 12 patients studied presented a Newton’s type 2 denture stomatitis and in every patient the same Candida species were isolated from the prosthesis and the palatal mucosa in contact with the dental prosthesis. CHROMagar Candida and the germ tube test allowed the differentiation of isolates giving green colonies and a positive germ tube test from those giving violet colonies and a negative germ tube test. Only the isolate from patient 8 was stained by the C. dubliniensis antiserum and showed abundant chlamydospore production on Casein agar. Eight isolates did not grow at 45ºC. Identification of all isolates was obtained by the ID 32C test. C. albicans was identified in 75% of patients, C. glabrata in 16,6% and C. dubliniensis in 8,3%. By using specific primers for typing C. dubliniensis, PCR allowed the identification of patient’s 8 isolate as C. dubliniensis genotype 1.
Conclusion: C. dubliniensis can be isolated from the oral cavity of teenagers wearing orthopedic oral prostheses and it is possible and technically amenable, the differentiation between C. albicans y C. dubliniensis using the ID 32C test, the observation of abundant chlamydospore production on Casein agar, the reactivity with a C. dubliniensis antiserum and the PCR.

Key words: Candida dubliniensis, denture stomatitis, Candidiasis

 

INTRODUCTION

Oral candidiasis is one of the most prevalent clinical presentations of Candida infections, particularly in human immunodeficiency virus (HIV)-infected individuals and other immunodeficient patients (1). In patients with a number predisposing conditions such as dental prosthesis, xerostomia, multiple therapies with antibiotics and immunosupressors, and some immunodeficiencies, member of genus Candida can convert into a pathogen capable of causing a variety of oral infections including pseudomembranous candidiasis, erythematous candidiasis and hyperplasic candidiasis, as well as Candida-associated denture stomatitis, Candida-associated angular cheilitis, rhomboid glossitis, exfoliative cheilitis and chronic mucocutaneous candidiasis. Candida may also be implicated in the linear gingival erythema and necrotic periodontitits in HIV-infected patients (1).

Although Candida albicans remains the most common cause of oral candidiasis, the incidence of disease caused by other species such as Candida krusei, Candida tropicalis, and Candida glabrata, has been increasing steadily (2, 3). The reason for the epidemiological change that is causing this emergence is not clear, although it has been suggested that the reduced susceptibility of these species to commonly used antifungal agents, such as fluconazole, may have led to their selection (4).

Candida dubliniensis is one of the emergent species which appeared to be mainly associated with oral disease in HIV-infected individuals (2, 5, 6) but it has also been recovered from other anatomical sites, from healthy individuals and in cases of systemic disease (2, 7-9). The very close phenotypic and genotypic relationship between C. dubliniensis and C. albicans has led to the misidentification of isolates of C. dubliniensis as C. albicans (2, 5), hampering comprehensive epidemiological analysis of this species (10). In recent years a number of phenotype-based and molecular tests have been developed which have permitted the rapid and reliable identification of C. dubliniensis in clinical samples (11-16). In the present study, we have used several methods that allow for the differentiation between C. albicans and C. dubliniensis in an attempt to assess whether C. dubliniensis can be recovered from the oral cavity of teenagers wearing orthopedic oral prostheses.

MATERIALS AND METHODS

Patients. Twenty-four oral specimens from 12 unrelated patients attending de Hospital Nacional de Odontología (Buenos Aires, Argentina) were studied. Oral examinations and clinical histories were performed by the same odontologist. Patients had an age range of 12-18, wore orthopedic dental prosthesis and had a non-relevant medical-odontological history. Exclusion criteria included immunosuppression, and existence of lesions associated with leukoplakia, erosive lichen and epitheliomas.

Oral swabs were obtained from the prosthesis as well as from the palatal mucosa in contact with the dental prosthesis.

Culture and identification. Clinical samples were grown on Sabouraud glucose agar plates (Difco, Detroit, Mich. USA) for 48 h at 30ºC. Control strains for identification tests included C. albicans serotype A (NCPF 3153) and C. dubliniensis CD36 (NCPF 3949, genotype 1) obtained from the National Collection of Pathogenic Fungi (NCPF, Bristol, United Kingdom) and C. albicans serotype B (ATCC 90028) obtained from the American Type Culture Collection (ATCC, Manassas, VA). For the typing of C. dubliniensis strains CBS 2747 (genotype 2) from Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands), p6265 (genotype 3) and p7718 (genotype 4) were also used (17). Conventional morphologic and physiologic methods including Gram staining, germ tube test, growth on CHROMagar Candida (CHROMagar Microbiology, Paris, Francia) and carbon assimilation by the ID 32C (bioMérieux, Marcy l’Etoile, Francia) (18), were employed to confirm the identity of all isolates. Serotyping of C. albicans isolates was performed according to Barturen et al. (19).

Differentiation between C. albicans and C. dubliniensis was achieved by a number of phenotypic tests and the polymerase chain reaction (PCR). Phenotypic tests included growth at 45ºC for 48 h on Sabouraud glucose agar (12), abundant chlamydospore production on Casein agar (14), and reactivity with a C. dubliniensis antiserum by indirect immunofluorescence (15). Identification and characterization of C. dubliniensis genotypes was performed by PCR with the primers G1F/G1R, G2F7/G2R, G3F/G3R and G4F/G4R, essentially as described by Brena et al. (17).

RESULTS

All 12 patients studied presented a Newton’s type 2 denture stomatitis (hyperhemic, smooth and atrophic) and yielded yeast colonies on Sabouraud’s dextrose agar. Identification of isolates showed that in every patient the same Candida species were isolated from the prosthesis and the palatal mucosa in contact with the dental prosthesis.

Careful observation of the colony color in CHROMagar Candida allowed the differentiation of isolates giving green colonies from those giving violet colonies (Table 1). The same differentiation was achieved with the germ tube test, since isolates producing green colonies had a positive germ tube test and those yielding violet colonies had a negative germ tube test. As it is difficult to differentiate C. albicans from C. dubliniensis by the colony color, and taking into consideration that both species can not be differentiated by the germ tube test, isolates producing green colonies and a positive germ tube test were identified as C. albicans/C. dubliniensis, while isolates producing violet colonies were identified as C. glabrata/C. parapsilosis/C. guilliermondii. Differentiation between C. albicans and C. dubliniensis was achieved by a number of phenotypic and genotypic tests. Phenotypic tests included abundant chlamydospore production on Casein agar, reactivity with a C. dubliniensis antiserum, growth at 45ºC and the ID 32C test. Only the isolate from patient 8 was stained by the C. dubliniensis antiserum and showed abundant chlamydospore production on Casein agar (Figure 1a and b, Table 1). Eight isolates did not grow at 45 ºC. Identification of all isolates was obtained by the ID 32C test. C. albicans was identified in 75% of patients, C. glabrata in 16,6% and C. dubliniensis in 8,3% (Table 1). The 48 h ID 32C code for patient’s 8 isolate was 7142140015+, allowing the identification of C. dubliniensis with a probability of 99,4%. All isolates identified as C. albicans/C. dubliniensis belonged to serotype A (Table 1).

By using specific primers for typing C. dubliniensis, PCR allowed the identification of patient’s 8 isolate as C. dubliniensis genotype 1 (Figure 1c).

DISCUSSION

Candida-associated denture stomatitis is an inflamatory process that affects the oral mucosa of 25-65% of patients wearing removable dental prostheses (20). Although denture stomatitis is usually diagnosed in adults, it can also be observed in children and teenagers with orthopedic dental prostheses (21). C. albicans is the major cause of fungal origin in denture stomatitis but other Candida species may also be involved (22). In this study C. albicans was isolated from the majority of patients with denture stomatitis but C. glabrata and C. dubliniensis were also isolated.As new studies are being performed, C. dubliniensis is being associated with an increasing number of clinical presentations of oral candidiasis. Although the most frequent clinical presentation of oral candidiasis caused by C. dubliniensis is erythematous candidiasis (23), cases of linear gingival erythema in children and denture stomatitis in adults caused by C. dubliniensis have also been described. However, in the denture stomatitis, C. dubliniensis was co-isolated with other Candida species (2, 24), and its participation in the pathogenesis of infection was not clear. To our knowledge, the patient described in this study is the first case of denture stomatitis caused directly by C. dubliniensis in a teenager wearing an orthopedic oral prosthesis. The clinical presentation of oral candidiasis in this patient, who wore an orthodontic appliance to increase the transversal diameter of the upper jaw, was not different from those observed in the rest of patients.

Differentiation between C. albicans and C. dubliniensis needs special tests, since conventional tests used in Mycology laboratories are not usually discriminatory. In this study, neither colony color on CHROMagar Candida nor lack of growth at 45 ºC were able to identify the C. dubliniensis isolate. The ID 32C test, the observation of the abundant chlamydospore production on Casein agar, the reactivity with a C. dubliniensis specific antiserum and the PCR were the only tests which allowed the differentiation between C. dubliniensis and C. albicans. The ID 32C test and the observation of the abundant chlamydospore production on Casein agar can be performed easily in the Mycology laboratories since the ID 32C is commercially available and Casein agar does not need sophisticated materials (14). However, although both tests allowed the identification of C. dubliniensis, the ID 32C test has been reported to be unable to identify all isolates of C. dubliniensis (11) and the presumptive identification obtained with the production of abundant chlamydospore production on Casein agar should be confirmed by other technique. In addition to the identification of C. dubliniensis, the ID 32C test allowed the accurate identification of C. albicans and C. glabrata isolates.

PCR analysis yielded two types of information. Firstly, it confirmed the identification of patient’s 8 isolate as C. dubliniensis, since the primers used did not amplify DNA from C. albicans isolates (17). Secondly, it was used to obtain the genotype, an information of epidemiological value. C. dubliniensis isolate from patient 8 belonged to genotype 1, which is the most frequent (25) and it has been predominantly detected in oral isolates in Spain (17).

In conclusion, results presented in this study demonstrate that C. dubliniensis can be isolated from the oral cavity of teenagers with denture stomatitis associated to orthopedic oral prostheses and that it is possible and technically amenable, the differentiation between C. albicans y C. dubliniensis using the ID 32C test, the observation of abundant chlamydospore production on Casein agar, the reactivity with a C. dubliniensis antiserum and the PCR.

ACKNOWLEDGEMENTS
This work was supported by grants 9/UPV 0093.327-13550/2001 from the Universidad del País Vasco and IE019, subproject Diamolfun, from the Departamento de Industria, Comercio y Turismo del Gobierno Vasco.

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