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Isolation, speciation and antifungal susceptibility testing of Candida isolates from various clinical specimens at a tertiary care hospital, Nepal

Background Candida species are responsible for various clinical infections ranging from mucocutaneous infection to life threatening invasive diseases along with increased resistance to antifungal drugs has made a serious concern. Resistance to antifungal agents has increased during the last decade. Thus, identification of Candida up to species level and its antifungal susceptibility testing has a paramount significance in the management of Candidal infections. The aim of the study was to speciate Candida species and to determine antifungal susceptibility pattern of Candida species to antifungal agents. Methods A total of 100 consecutive Candida species were isolated from 1248 clinical specimens over 7 months period. Growths on Sabouraud dextrose agar were evaluated for colony appearance, macroscopic examination, Gram staining, germ tube test and urea hydrolysis test. Further, they were processed for Candida speciation on CHROMagar. Antifungal susceptibility testing was performed as recommended by Clinical and Laboratory Standards Institute (CLSI) M44-A document. Results Out of 100 Candida isolates, Candida albicans (56%) was the most common species. Among the non-albicans Candida species, Candida tropicalis (20%) was the predominant isolate followed by Candida glabrata (14%). Regarding antifungal susceptibility pattern, Candida species were more susceptible to clotrimazole (82%) followed by fluconazole (64%) and miconazole (44%). Conclusions Candida albicans was the predominant species responsible for various Candidal infections. Among commonly used antifungal drugs clotrimazole, miconazole and fluconazole were most effective.


Background
Candida species is a normal commensal flora of human body inhabiting skin, mucous membranes and gastrointestinal tract but may be associated with superficial and deep seated fungal infections [1]. The switch of Candida species from commensal to a potent pathogen is facilitated by various virulence factors such as adherence to host tissues, medical devices, biofilm formation, and secretion of extracellular hydrolytic enzymes [2]. Also, in recent year non-albicans Candida (NAC) species are considered as major pathogens causing severe infections in human [3].
The commonly used antifungal drugs show significant variation in the susceptibility pattern among the types of Candida species. The drug resistance scenario has been Open Access BMC Research Notes *Correspondence: cls.sundar@iom.edu.np; cls.sundar@gmail.com 1 HIV Reference Unit, National Public Health Laboratory, Kathmandu, Nepal Full list of author information is available at the end of the article increasing during last decades due to over growing use of random antifungal agents [4]. Several previous studies reported the emergence of drug resistance Candida species in global scenario [5,6]. Therefore, the change in drug susceptibility pattern of Candida species and introduction of newer antifungal agents has made the in vitro susceptibility testing of antifungal agents more relevant for using specific and sensitive drugs.
Thus, the isolation, identification, characterization and susceptibility testing of Candida species in clinical specimens have become increasingly important for management of fungal infections. CHROMagar medium is an easy, rapid and reliable method to isolation and for differentiation of four types of Candida species [7].
In the present study, we explored the characterization of Candida species using CHROMagar and showed the susceptibility pattern of Candida isolates from clinical specimens.

Methods
A laboratory based cross sectional study was carried out in the Department of Microbiology, Tribhuvan University Teaching Hospital from July 2014 to January 2015. A total of 1248 different clinical specimens (urine, sputum, catheter tip, blood, high vaginal swabs and endotracheal tube) proceeded for laboratory investigation. The preliminary diagnoses of specimens were performed by wet mount, Gram stain, culture on Sabouraud dextrose agar (SDA) and urea hydrolysis test. The isolates diagnosed to be fungus other than Candida species were excepted from the study. For the clinical significance of Candida isolates from sputum and urine, the specimens were analysed by microscopy as well for the evidence of budding yeast cell with pseudohyphae along with significant pus cells [8,9]. All samples were inoculated on Sabouraud dextrose agar (SDA) slants supplemented with chloramphenicol and aerobically incubated at 37 °C for 24-48 h. For blood culture, 8-10 ml venous blood was collected aseptically and cultured in 45 ml Brain heart infusion (BHI) broth. It was then incubated at 37 °C for up to 96 h before reported as no growth.
Any visible growth seen on SDA slope was processed for identification of the species. From an isolated colony, macroscopic examination, Gram staining, germ tube test and urea hydrolysis test was performed. The yeasty, pasty and creamy colony that showed Gram positive budding yeast cells with pseudohyphae on microscopic examination and negative urea hydrolysis test were further processed for Candida speciation on CHROMagar. Candida species were differentiated based on type of the growth and colour of isolates on CHROMagar Candida (HiMedia, Mumbai, India) [10,11]. After incubation at 37 °C for 24-48 h, colour of colonies was observed on CHROMagar (C. albicans-light green, C. glabratacream to white, Candida krusei-purple, fuzzy and C. tropicalis-blue to purple).
Antifungal susceptibility testing was performed and interpreted for all the isolates of Candida using disc diffusion method as recommended by Clinical and Laboratory Standards Institute (CLSI) M44-A document guidelines [12]. The inoculum was prepared by suspending five colonies of growth in 5 ml of sterile saline and compared the turbidity to 0.5 McFarland Standard. A cotton swab was dipped into the inoculum suspension and evenly streaked onto Mueller-Hinton agar supplemented with 2% glucose and 5 µg/ml methylene blue [13,14]. C. albicans ATCC 90028, C. tropicalis ATCC 750 were used as controls.

Results
A total of 100 Candida species were isolated from urine (48%), sputum (42%), catheter tip (4%), blood (2%), high vaginal swabs (2%) and endotracheal tube (2%) as shown in Table 1. We analysed 1248 clinical specimens and that showed 8.1% culture positivity. Gender-wise distribution showed that 52% Candida isolates were from male and 48% from female. Based on CHROMagar, four types of Candida species were differentiated. C. albicans (56%) was the most frequently encountered species and the bulk of isolates were from urine and sputum samples (26 and 24% respectively). Among the NAC spp., C. tropicalis (20%) was the commonest isolate followed by C. glabrata (14%) and C. krusei (10%) respectively. All four types of Candida species were isolated from urine samples whereas C. albicans was isolated from all specimens except the endotracheal tube. The different species of Candida reported from various specimens in our study is showing in Table 1.
Overall, antifungal susceptibility profile of Candida species to ketoconazole was found to be 6% susceptible (S), 8% susceptible dose dependent (SDD) and 86% resistant (R). In similar way, antifungal profile (S, SDD, R) to fluconazole was (64, 16 and 20% respectively), to miconazole (44, 44, 12%) and to clotrimazole (82, 12, 6%) as depicted in Table 2. Among the four antifungal agents, the highest level of susceptibility was observed in clotrimazole, followed by fluconazole and miconazole respectively whereas ketoconazole showed the highest level of resistance.
In this study, C. albicans (89.3%) was found more resistant to ketoconazole with compared to NAC spp. Among the NAC spp., C. krusei showed 20% sensitivity whereas no sensitive results were observed in C. tropicalis and C. glabrata. In fluconazole, more resistance was observed in C. glabrata (42.9%), whereas no resistance was observed for C. krusei. C. albicans isolates (71.5%) were more sensitive to fluconazole with compared to NAC spp.
In miconazole, more resistance was observed for C. krusei (20%) while no resistance was observed for C. glabrata. C. albicans isolates (53.6%) were more sensitive to miconazole compared to NAC spp. In clotrimazole, more resistance was observed in C. glabrata (14.2%), no resistance was observed in C. tropicalis and C. krusei. All isolates of C. krusei were found to be sensitive to clotrimazole. Antifungal susceptibility profile of different Candida species to antifungal agents is showing in Table 2.

Discussion
In this study, a total of 100 Candida isolates were obtained over 7 months of period from different clinical specimens. The majority of Candida species were isolated from urine and sputum that cover 90%, which indicates the higher incidence and distribution of Candida species causing urinary tract and respiratory tract infections.
Of the 100 Candida isolates, C. albicans was the predominant species (56%) followed by C. tropicalis (20%), C. glabrata (14%) and C. krusei (10%) respectively. Our finding shows similar prevalence scenario of Candida species and susceptibility pattern to the previous data reported by two independent groups from India which showed C. albicans is more prevalent among the Candida isolates [16,17]. Similar study conducted by Sajjan et al. also reported C. albicans as the major isolate. Among the NAC species, C. tropicalis was most prevalent followed by C. glabrata and C. krusei respectively [18]. Jayalakshmi et al. also showed that C. tropicalis (26.6%) was prevalent among the NAC species [19]. Similar result has been depicted in various studies conducted in different countries of Europe [20][21][22]. However, many studies have shown that NAC species have more isolation rate than C. albicans which suggest the emergence of non-albicans Candida species as important pathogens [23,24]. Speciation of Candida species by CHROMagar on the basis of colour differentiation offered a rapid, convenient and reliable method for identification of clinically important Candida species when compared with cumbersome traditional techniques. In developing countries, CHRO-Magar can be taken as a simple phenotypic test alternative to molecular based assay. CHROMagar has high sensitivity as well as specificity for the identification of Candida species [10,25]. According to various finding from our regions, these four species are more prevalent, so we chose this medium for isolation of Candida spp. [17,26]. It facilitates the detection and identification of Candida species from mixed culture and provides results within 24-48 h.
In this study, Candida species were found to be more susceptible to clotrimazole (82%) followed by fluconazole (64%) and miconazole (44%) respectively whereas 86% of the isolates were resistant to ketoconazole. Among the four antifungal agents, clotrimazole and miconazole had not been used for clinical treatment but might be of interest for mycological study. A similar study was conducted to perform an antifungal test to clotrimazole and miconazole as of mycological interest [27,28]. In the present study, 20% of total isolates were found to be resistant to fluconazole by disc diffusion method. The highest rate of fluconazole resistance was observed in C. glabrata (42.9%), followed by C. tropicalis (20%) and C. albicans (17.9%). Our finding is very close with the findings of Mondal et al., which also showed 18% Candida spp. and 19.2% C. tropicalis were resistance to fluconazole. On the contrary, all C. krusei isolates were sensitive to fluconazole in our study while 60% were found to be susceptible dose dependent for the same. In a case of C. krusei, some degree of susceptibility was seen to fluconazole, comparable to other studies by applying same interpretative criteria as introduced by CLSI [15,29]. However, it is reported as intrinsically resistant to fluconazole. So, it showed further investigation is needed for solving the query of fluconazole sensitivity. The increase in resistance to fluconazole is of serious concern as it is the most commonly used azole for superficial as well as deep candidiasis.
This study also reveals the higher resistant rate for ketoconazole (86%). This high level of resistance of ketoconazole might be due to overuse of antifungal agents and also their empirical therapy in our scenario. The higher rate of ketoconazole resistance was seen in C. albicans (89.3%), C. glabrata (85.8%), and followed by C. tropicalis and C. krusei (80% for both). Our result hugely differs from the findings by Binesh et al. in which only 2.1% C. albicans isolates were resistant to ketoconazole [30]. Furthermore, Mondal et al. revealed overall 11.7% resistance to ketoconazole and with Candida krusei (20%) followed by C. glabrata (17.6%), C. tropicalis (15.2%) and least being C. albicans (7.8%). The study conducted by Zomorodian et al. showed that fluconazole sensitivity was seen in 96.6% of the Candida isolates [31].
These findings suggest the rapid increase in resistance among Candida species for ketoconazole and need for speciation and antifungal susceptibility before treatment with antifungal drug.

Conclusions
In our study we found that C. albicans was the predominant species responsible for various Candidal infections. Among commonly used antifungal drugs clotrimazole, miconazole and fluconazole demonstrated a high rate of sensitivities while ketoconazole was the least effective for both C. albicans and NAC spp. Species identification of Candida has a paramount effect on successful treatment as it helps in optimum selection of the therapeutic agent and use of CHROMagar is a simple, rapid and inexpensive method for identification of Candida species especially in the laboratory with limited resources.
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