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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 4  |  Page : 501-504
Detection and antifungal susceptibility testing of oral Candida dubliniensis from human immunodeficiency virus-infected patients


1 Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad-580 009, India
2 Department of Microbiology, Karnataka Institute of Medical Sciences, Hubli- 580022, India
3 Department of Medicine, SDM College of Medical Sciences and Hospital, Dharwad-580 009, India
4 Department of Microbiology, Al Ameen Medical College, Bijapur- 586108, India
5 Department of Microbiology, Raichur Institute of Medical Sciences, Raichur- 584102, India

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Date of Web Publication1-Oct-2009
 

   Abstract 

Context: Candida dubliniensis, an opportunistic yeast that has been implicated in oropharyngeal candidiasis (OPC) in patients infected with Human Immunodeficiency Virus (HIV) may be under-reported due to its similarity with Candida albicans. Resistance to Fluconazole is often seen in C. dubliniensis isolates from clinical specimens. Aims: To know the prevalence of C. dubliniensis in OPC in patients infected with HIV and their antifungal susceptibility pattern. Settings and Design: One hundred and thirty-two HIV seropositive individuals and 50 healthy controls were included in the study. Materials and Methods: Two oral swabs were collected from the site of the lesion from 132 HIV-infected patients. Oral rinse was obtained from 50 healthy controls. Samples were inoculated on Sabouraud's dextrose agar (SDA) medium and on HiCrome Candida Differential Agar (CHROM agar) medium. Isolates were speciated by standard tests. Dark green-colored, germ tube positive isolates, which failed to grow at 420C and negative for xylose assimilation were identified as
C. dubliniensis. Antifungal susceptibility test was performed by Macro broth dilution technique (National Committee for Clinical Laboratory Standards guidelines). Results and Conclusions: From 132 patients, 22 (16.3%) C. dubliniensis were isolated; samples from healthy controls did not reveal their presence. Antifungal susceptibility test showed higher resistance among C. dubliniensis isolates to azoles compared to C. albicans. Five (22.7%) isolates of C. dubliniensis were resistant to Fluconazole followed by four (18.2%) to Ketoconazole. This study emphasizes the importance of identification and antifungal susceptibility testing of C. dubliniensis in HIV-infected patients.

Keywords: Antifungal susceptibility testing, C. dubliniensis, human immunodeficiency virus, oropharyngeal candidiasis

How to cite this article:
Chunchanur SK, Nadgir SD, Halesh L H, Patil B S, Kausar Y, Chandrasekhar M R. Detection and antifungal susceptibility testing of oral Candida dubliniensis from human immunodeficiency virus-infected patients. Indian J Pathol Microbiol 2009;52:501-4

How to cite this URL:
Chunchanur SK, Nadgir SD, Halesh L H, Patil B S, Kausar Y, Chandrasekhar M R. Detection and antifungal susceptibility testing of oral Candida dubliniensis from human immunodeficiency virus-infected patients. Indian J Pathol Microbiol [serial online] 2009 [cited 2014 Jul 28];52:501-4. Available from: http://www.ijpmonline.org/text.asp?2009/52/4/501/56138



   Introduction Top


Oropharyngeal candidiasis (OPC) is the most common opportunistic infection in the immunocompromised. Although C. albicans is a well-known etiological agent of OPC, C. dubliniensis has emerged as another pathogen known for its azole resistance. But as C. dubliniensis phenotypically resembles C. albicans very closely, it may be misidentified. [1]

Continuing importance of C. dubliniensis as a significant opportunistic pathogen and its predilection for Fluconazole resistance makes it imperative for the laboratories to identify C. dubliniensis correctly. [2] Isolation of C. dubliniensis for the first time in India was reported in 2003. [3] In this study an attempt has been made to know the prevalence and antifungal susceptibility pattern of C. dubliniensis in HIV-infected patients with OPC.


   Materials and Methods Top


Clinical samples were obtained from HIV-infected patients admitted in the hospital after taking informed consent. Two oral swabs were collected from the site of the lesion from 132 HIV-infected patients presenting with oral lesions suggestive of candidiasis. Oral rinse was obtained from 50 non-HIV-infected healthy individuals without any oral lesion to know the prevalence of colonization of candida in the healthy population. Samples were inoculated on Sabouraud's dextrose agar (SDA). Additionally, samples were also inoculated on HiCrome Candida Differential Agar (CHROM agar) (Hi Media Pvt Ltd, Mumbai) to improvise species identification, based on colored colony morphology. SDA slants and CHROM agar plates were incubated at 37 0 C for 48 h. Isolates were further characterized by performing the following tests: Germ tube production, chlamydospore formation on corn meal agar, sugar assimilation profile and differential growth on SDA at 37 0 C and 42 0 C. [4],[5],[6]

Isolates producing dark green-colored colonies on CHROM agar, which were also germ tube-positive, chlamydospore-producing, but failed to grow at 42 0 C and failed to assimilate xylose were phenotypically identified as C. dubliniensis.[4],[5],[6],[7]

Antifungal susceptibility testing of the isolates for Amphotericin B, Ketoconazole and Fluconazole was performed by Macro broth dilution technique according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Minimum Inhibitory Concentration (MIC) was calculated for each isolate, based on which isolates were interpreted to be sensitive, intermediate and resistant. [8]


   Results Top


Candida was recovered from 132 HIV seropositive patients having oral lesions suggestive of OPC. Samples from three patients revealed a mixture of two species, making a total of 135 candida isolates from 132 patients. Candida was isolated only from three non-HIV-infected healthy controls. As per the World Health Organization (WHO) staging system of HIV infection, most of the HIV positive patients included in our study belonged to category 3C.

On CHROM agar medium 90 (66.6%) isolates produced light green colonies and 22 (16.3%) produced dark green-colored colonies. One hundred and twelve (83%) isolates were germ tube-positive and also formed chlamydospores on corn meal agar. Of which, 90 (66.6%) isolates assimilated dextrose, maltose, sucrose, galactose and xylose which is typical of C. albicans. Twenty-two (16.3%) isolates produced similar assimilation pattern except for xylose, which was not assimilated. Growth at differential temperature of 37 0 C and 42 0 C on SDA after 48 h incubation revealed that 22 (16.3%) isolates failed to grow at 42 0 C. Based on these findings, 22 (16.3%) isolates producing dark green colonies on CHROM agar, germ tube-positive, chlamydospore-forming, which failed to grow at 42 0 C were phenotypically identified as C. dubliniensis. All the three isolates from healthy controls were C. albicans.

Antifungal susceptibility testing of C. dubliniensis [Table 1] showed that all 22 (100%) isolates were susceptible to Amphotericin B, with an MIC range of 0.03-0.5 μg/ ml. Only 12 (54.6%) isolates were sensitive to Fluconazole with an MIC range of 0.125-0.5 μg/ml. Against Ketoconazole, 14 (63.6%) isolates showed sensitivity with MIC range of 0.03-0.5 μg/ml. High-level resistance to azoles was observed among C. dubliniensis isolates, five (22.7%) isolates were resistant to Fluconazole with MIC range of 16-64 μg/ml and five (22.7%) were intermediate (dose-dependent) sensitive with MIC of 1-8 μg/ml. Whereas, against Ketoconazole, four (18.2%) were resistant, with MIC range of 8-16 μg/ml and four (18.2%) isolates showed dose-dependent sensitivity with MIC range of 1-4 μg/ml. A very high MIC of >32 μg/ml was noted among three isolates of C. dubliniensis resistant to Fluconazole. But among isolates of C. albicans, not only were all found to be sensitive to Amphotericin B, resistance to Azoles was also found to be low. Only 11 (12.2%) isolates of C. albicans showed resistance to Fluconazole and nine (10%) against Ketoconazole.

[TAG:2]Discussion[/TAG:2] In the present study prevalence of C. dubliniensis was 22 (16.3%) amongst 135 candida strains isolated. Various reports from different parts of the world have quoted prevalence ranging from 16 to 32%. [9]

C. dubliniensis has been differentiated from C. albicans in our study, based on colony morphology on CHROM agar medium, chlamydospore formation, xylose assimilation and growth at differential temperature. All 22 (16.3%) isolates produced dark green colonies on CHROM agar medium, did not assimilate xylose and failed to grow at 42 0 C. Abundant chlamydospore production could not be appreciated in all; moreover, comparison of relative abundance can be subjective.

C. albicans and C. dubliniensis can be differentiated from all other candida species by their ability to produce germ tubes and chlamydospores. It is not possible to distinguish between C. albicans and C. dubliniensis colonies on conventional solid media i.e. SDA. But the colonies of the two species can be distinguished following primary isolation from clinical specimens by using CHROM agar medium, which is useful only for identifying colonies of C. dubliniensis following primary plating from clinical specimens, because C. dubliniensis isolates lose their ability to yield dark green colonies on CHROM agar medium following subculture or storage. [10]

Regarding sugar assimilation profile, xylose is assimilated by C. albicans isolates but not by C. dubliniensis. Conventional sugar assimilation testing methods can be used for identifying C. dubliniensis routinely, [11] as use of automated systems such as API20 is unaffordable due to its high cost. [12]

Differential growth on SDA at 37 0 C and 42 0 C is extremely useful as a simple and inexpensive method for presumptive identification of C. dubliniensis, as at 42 0 C all C. albicans grow well, but C. dubliniensis either grows scantily or not at all at that temperature. [13]

Apart from conventional phenotypic methods used in this study for identification of C. dubliniensis, definitive identification can be obtained by testing for lack of ί galactosidase activity, lack of fluorescence under long-wave ultraviolet light following growth on methyl blue Sabouraud's agar. But the most pronounced differences are genetic, as determined by DNA fingerprinting, karyotype analysis and DNA sequence analysis of r RNA genes. However, the techniques used to detect these genetic differences are time-consuming and are not readily applicable to a large number of samples. [6],[12] To be of use identification techniques must be inexpensive, easy to apply and reproducible.

Antifungal susceptibility testing revealed high-level resistance to azoles among C. dubliniensis isolates compared to C. albicans. Five (22.7%) isolates of C. dubliniensis were resistant to Fluconazole with MIC range of 16-64 μg/ml, followed by four (18.2%) to Ketoconazole with MIC range of 8-16 μg/ml.

There are reports of isolation of Fluconazole-resistant C. dubliniensis clinical isolates from oral cavities of HIV-infected patients with OPC prior to exposure to Fluconazole. In addition, derivatives exhibiting a stable Fluconazole-resistant phenotype can be readily generated in vitro from Fluconazole-susceptible isolates, a phenomenon that is not easily observed with C. albicans. However, isolates of C. dubliniensis resistant to Fluconazole may still be sensitive to other azole compounds such as Itraconazole and Voriconazole. [14]

Replacement of C. albicans by C. dubliniensis is known to occur in patients treated with Fluconazole. The antifungal pressure exerted by this drug influences the oral microbial ecology in these patients, as species that are better able to adapt to antifungal pressure persist over those that are suppressed by the treatment. [15]

Despite the close phenotypic resemblance between C. albicans and C. dubliniensis, discriminating between the two species in the clinical laboratory should no longer be a difficult task due to the availability of simple and rapid phenotypic tests which if used in combination and interpreted properly , provide reliable identification of C. dubliniensis.

The findings that a significant proportion of isolates had reduced susceptibility to Fluconazole as shown in our study and the fact that susceptible isolates of C. dubliniensis that were once susceptible may develop resistance in future may have implication for change in antifungal drug regimens for treatment of OPC in HIV infected individuals.

To conclude, proper identification of C. dubliniensis to know its prevalence in a particular geographical area and studying its antifungal susceptibility pattern is of importance for successful treatment of OPC in HIV seropositive individuals. [3] Phenotypic methods can provide accurate identification of C. dubliniensis. Also, the interaction and ecological relationship of different candida species in the oropharynx, especially C. dubliniensis, due to its predilection for azole resistance and increased adherence to buccal epithelial cells constitutes a topic of active research.

 
   References Top

1.Martinez M, Lopez-Ribot JL, Kirkpatrick WR, Coco BJ, Bachmann SP, Patterson TF. Replacement of Candida albicans with C. dubliniensis in Human Immunodeficiency Virus infected patients with oropharyngeal candidiasis treated with fluconazole. J Clin Microbiol 2002;40:3135-9.   Back to cited text no. 1      
2.Jabra-Rizk MA, Falkler WA Jr, Merz A, Baqui AAMA, Kelley JI, Meiller TF. Retrospective identification and characteristics of Candida dubliniensis isolates among Candida albicans clinical laboratory isolates from HIV infected and noninfected patients. J Clin Microbiol 2000;38:2423-26.   Back to cited text no. 2      
3.Gugnani HC, Becker K, Fegeler W, Basu S, Chattopadhya D, Baveja U, et al. Oropharyngeal carriage of Candida species in HIV-infected patients in India. Mycoses 2003;46:299-306.   Back to cited text no. 3      
4.Rippon JW. Candidiasis and the pathogenic yeasts. In: Martin Wonseiwicz editors. Medical Mycology. Philadelphia: W.B. Saunders; 1988. p. 531- 81.   Back to cited text no. 4      
5.Khan ZU, Ahmad S, Mokaddas E, Al-Sweih N, Chandy R. Sunflower seed husk agar: A new medium for the differentiation of Candida dubliniensis from Candida albicans. Indian J Med Microbiol 2005;23:182-5.  Back to cited text no. 5      
6.Marot-Leblond A, Grimaud L, David S, Sullivan DJ, Coleman DC, Jose Ponton, et al. Evaluation of a Rapid Immunochromatographic Assay for Identification of Candida albicans and Candida dubliniensis. J Clin Microbiol 2004;42:4956-60.  Back to cited text no. 6      
7.Sullivan D, Coleman D. Candida dubliniensis: Characteristics and identification. J Clin Microbiol 1998;36:329-34.  Back to cited text no. 7      
8.Lattif AA, Banerjee U, Prasad R, Biswas A, Wig N, Sharma N, et al. Susceptibility Pattern and Molecular Type of Species-Specific Candida in oropharyngeal lesions of Indian human immunodeficiency virus-positive patients. J Clin Microbiol 2004;42:1260-2.  Back to cited text no. 8      
9.Coleman DC, Sullivan DJ, Bennet DE, Moran GP, Barry HJ, Shanley DB. Candidiasis: The emergence of a novel species, Candida dubliniensis. AIDS 1997;11:557- 67.  Back to cited text no. 9      
10.Tintelnot K, Haase G, Seibold M, Bergmann F, Staemmler M, Franz T, et al. Evaluation of phenotypic markers for selection and identification of Candida dubliniensis. J Clin Microbiol 2000;38:1599-608.  Back to cited text no. 10      
11.Pincus DH, Coleman DC, Pruitt WR, Padhye AA, Salkin IF, Geimer A, et al. Rapid identification of Candida dubliniensis with commercial yeast identification systems. J Clin Microbiol 1999;37:3533-9.  Back to cited text no. 11      
12.de Loreto ES, Pozzatti P, Alves Scheid L, Santurio D, Morais Santurio J, Alves SH. Differentiation of Candida dubliniensis from Candida albicans on rosemary extract agar and oregano extract agar. J Clin Lab Anal 2008;22:172- 7.  Back to cited text no. 12      
13.Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis and Candida albicans. J Clin Microbiol 1998;36:2093-5.  Back to cited text no. 13      
14.Kirkpatrick WR, Revankar SG, Mcatee RK, Lopez-Ribot JL, Fothergill AW, McCarthy Di, et al. Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus- infected patients in North America by primary chromagar candida screening and susceptibility testing of isolates. J Clin Microbiol 1998;36:3007-12.   Back to cited text no. 14      
15.Hamza OJ, Matee MI, Moshi MJ, Simon EN, Mugusi F, Mikx FH, et al. Species distribution and in vitro antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis. BMC Microbiol 2008;8:135.  Back to cited text no. 15      

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Correspondence Address:
Sneha K Chunchanur
Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad-580009, Karnataka
India
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DOI: 10.4103/0377-4929.56138

PMID: 19805956

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