Antifungal resistance is a major concern in clinical practice. The advance of therapy has increased the survival rate but the risk factors have accumulated with an increase in cases of infectious diseases [1Spampinato C, Leonardi D. Candida infections, causes, targets, and resistance mechanisms: Traditional and alternative antifungal agents. BioMed Res Int 2013; 2013: 204237.
[http://dx.doi.org/10.1155/2013/204237] [PMID: 23878798] , 2Emerging Threats in Antifungal-Resistant Fungal Pathogens. Front Med 2016; 3: 10-1.]. In fungus, C. albicans is the most vital opportunistic pathogen where it normally resides in oral, conjunctival, gastrointestinal and genitourinary tracts Moreover, infection caused by Candida species is called as candidiasis [3Ako-Nai AK, Kassim OO, Adeniran MO, Taiwo O. A study of urinary tract infections at Ile-Ife, Nigeria: East. Afr Med 1993; 70: 110-4.]. About 75% women have this fungus without it causing infection [4Dan M, Poch F, Levin D. High rate of vaginal infections caused by non-C. albicans Candida species among asymptomatic women. Med Mycol 2002; 40(4): 383-6.
[http://dx.doi.org/10.1080/mmy.40.4.383.386] [PMID: 12230217] , 5Ellepola AN, Samaranayake LP. The effect of limited exposure to antimycotics on the relative cell-surface hydrophobicity and the adhesion of oral Candida albicans to buccal epithelial cells. Arch Oral Biol 1998; 43(11): 879-87.
[http://dx.doi.org/10.1016/S0003-9969(98)00064-8] [PMID: 9821511] ]. The factors that predispose women to vaginal candidiasis are changes in pH, use of oral contraceptives, tight clothing, and personal hygiene [6Carlsen G. The Candida Yeast Answer 2001., 7Enweani IB, Gugnani HC, Okobia R, Ojo SB. Effect of contraceptives on the prevalence of vaginal colonization with Candida species in Edo State, Nigeria. Rev Iberoam Micol 2001; 18(4): 171-3.
[PMID: 15496123] ].

There is an extensive use of fluconazole and itraconazole for chemoprophylaxis and treatment of fungal infections due to their favorable oral bioavailability and safety. Hence it has led to fluconazole resistance in a high percentage [8Redding SW, Kirkpatrick WR, Saville S, et al. Multiple patterns of resistance to fluconazole in Candida glabrata isolates from a patient with oropharyngeal candidiasis receiving head and neck radiation. J Clin Microbiol 2003; 41(2): 619-22.
[http://dx.doi.org/10.1128/JCM.41.2.619-622.2003] [PMID: 12574256] ]. Azole-resistant C. albicans is frequently observed in oropharyngeal candidiasis of HIV patients [9Skiest DJ, Vazquez JA, Anstead GM, et al. Posaconazole for the treatment of azole-refractory oropharyngeal and esophageal candidiasis in subjects with HIV infection. Clin Infect Dis 2007; 44(4): 607-14.
[http://dx.doi.org/10.1086/511039] [PMID: 17243069] ]. Apart from albicans, species of Candida like C. glabrata, C. krusei, and C. lusitaniae have been reported with reduced its susceptibility to fluconazole [10Vazquez JA, Peng G, Sobel JD, et al. Evolution of antifungal susceptibility among Candida species isolates recovered from human immunodeficiency virus-infected women receiving fluconazole prophylaxis. Clin Infect Dis 2001; 33(7): 1069-75.
[http://dx.doi.org/10.1086/322641] [PMID: 11528582] , 11Safdar A, van Rhee F, Henslee-Downey JP, Singhal S, Mehta J. Candida glabrata and Candida krusei fungemia after high-risk allogeneic marrow transplantation: No adverse effect of low-dose fluconazole prophylaxis on incidence and outcome. Bone Marrow Transplant 2001; 28(9): 873-8.
[http://dx.doi.org/10.1038/sj.bmt.1703252] [PMID: 11781648] ]. Pfalleret al., 2004 reported that changes in the structure of azoles are the factors responsible for cross-resistance patterns among Candida species. Moreover the environmental stress with exposure to antifungal drugs can mediate resistance [13Zaidi KU, Mani A, Thawani V, Mehra A. Total protein profile and drug resistance in candida albicans isolated from clinical samples. Mol Biol Int 2016; 2016: 4982131.
[http://dx.doi.org/10.1155/2016/4982131] [PMID: 27478638] ]. The present study evaluates the prevalence of antifungal resistance in C. albicans isolated from catheter tip, oral, urine and high vaginal swab. Where, antifungal resistance against amphotericin B, ketoconazole, itraconazole and fluconazole was analyzed. The antifungal resistance against infected location was correlated to visualize the development of drug resistance and to estimate the prevalence of resistant strains towards the site of infection which will help to known the sensitive site procedure. The study was conducted to assess the C. albicans management, as it is one of the normal human floras and its alternation to antifungal resistance is a concern for human health with the development of drug resistance.

In this study, 200 clinical C. albicans were isolated; of which 10.6% were of high-risk patients. The age group of patients in the study was from 18 to 90 years. Slight female preponderance was observed in the study with M: F ratio of 0.86:1.14.The number of C. albicans isolates (Table 1) ranged from U (20%), HVS (22.5%) CT (22.5%), O (22.5%) to B (12.5%). It was observed that there is considerable variation among C.albicans between the site and patients. It is also reported a high degree of variability among their isolates [16Merz WG, Connelly C, Hieter P. Variation of electrophoretic karyotypes among clinical isolates of Candida albicans. J Clin Microbiol 1988; 26(5): 842-5.
[PMID: 3290238] ].

Table 1
Antifungal sensitive patternwith respect to the rute of pathogen.

The clinical isolates were identified up to the genus level. Hyphal morphology of isolates and characteristics of the spores were observed using lactophenol cotton blue stain under the light microscope. Morphological and physiological characteristics of the isolates were used for the identification of yeasts based on the Buschelman et al. [17Buschelman B, Jones RN, Pfaller MA, Koontz FP, Doern GV. Colony morphology of Candida spp. as a guide to species identification. Diagn Microbiol Infect Dis 1999; 35(1): 89-91.
[http://dx.doi.org/10.1016/S0732-8893(99)00051-6] [PMID: 10529886] ], morphological identification of Candida spp.

In the present study, we examined the antifungal susceptibility of antifungal agents. Using C. albicans in disk diffusion and a micro-dilution method, comparative efficacies of ketoconazole, amphotericin B, fluconazole and itraconazole were examined in vitro. The zone of inhibition of a different antifungal agent against C. albicans (mean±SD) was observed at a concentration of 100µg/ml (Fig. 1).The susceptibility pattern of the isolates showed 100% sensitivity to ketoconazole and amphotericin-B, moreover Fluconazole (81 isolates) and itraconazole (63 isolates) resistances were observed. Antifungal susceptibility test revealed that all isolates (200) were sensitive to amphotericin-B and ketoconazole. However, significant resistance of 56.5% and 64.5% was observed for fluconazole and itraconazole, respectively. It was observed that 158 strains were sensitive and 42 strains were resistant to fluconazole and itraconazole. CT showed high resistant strains being 42.2% followed by HVS strains. Strains isolated from blood were sensitive to both the antibiotics (Table 1).Our findings are in accordance with the study conducted by Fadda et al.,2008 [18Fadda ME, Podda GS, Pisano MB, Deplano M, Cosentino S. Prevalence of Candida species in different hospital wards and their susceptibility to antifungal agents: Results of a three year survey. J Prev Med Hyg 2008; 49(2): 69-74.
[PMID: 18847180] ] where decreased susceptibility to azoles in C. albicans. was observed. It is also reported similar susceptibility of C. albicans [19Hadley S, Martinez JA, McDermott L, Rapino B, Snydman DR. Real-time antifungal susceptibility screening aids management of invasive yeast infections in immunocompromised patients. J Antimicrob Chemother 2002; 49(2): 415-9.
[http://dx.doi.org/10.1093/jac/49.2.415] [PMID: 11815592] ].

Fig. (1) Antifungal susceptibility in Candida albicans; [A] Oral, [B] Catheter tip, [C] High vaginal swab, [D] Urine, [E] Blood.

Minimum inhibitory concentration was tested at the final concentrations ranging from0.5 μg/ml to 64μg/ml. The wells that showed no color (pink) of the reagent (MTT) indicate the concentration at which the fungal growth was inhibited and the lowest concentration showing no colour was recorded in terms of the MIC value. The MIC values of fluconazole for the isolates ranged from 16μg/ml, 32μg/ml to 64μg/ml with 18.63%, 32.4% and 27.5% susceptibility, respectively. In case of itraconazole, the isolates showed susceptibility of 30.9%, 11.34%and 19.53% towards 16, 32 and 64μg/ml of antibiotic, respectively. Moreover, all isolates were observed to be sensitive for ketoconazole and amphotericin-B with 25% and 60% at 0.5μg/ml and 75% and 40% at 1μg/ml susceptibility, respectively (Table 2). Early detection of drug susceptibility to the organism has to be carried out for a successful treatment of any infectious disease [20Hospenthal DR, Murray CK, Rinaldi MG. The role of antifungal susceptibility testing in the therapy of candidiasis. Diagn Microbiol Infect Dis 2004; 48(3): 153-60.
[http://dx.doi.org/10.1016/j.diagmicrobio.2003.10.003] [PMID: 15023422] , 21Meletiadis J, Melchers WJ, Meis JF, Van Den Hurk P, Jannes G, Verweij PE. Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids. Med Mycol 2003; 41(1): 65-74.
[http://dx.doi.org/10.1080/mmy.41.1.65.74] [PMID: 12627806] ]. In immunodeficient or immune suppressed patients, early therapies can alter the course of fungal infections. It was stated that antifungal susceptibility testing is useful to know the susceptibility in correlation with 48- and 24-h fluconazole MICs in Candida isolates showed good correlation to visual MICs [12Pfaller MA, Messer SA, Boyken L, et al. Cross-resistance between fluconazole and ravuconazole and the use of fluconazole as a surrogate marker to predict susceptibility and resistance to ravuconazole among 12,796 clinical isolates of Candida spp. J Clin Microbiol 2004; 42(7): 3137-41.
[http://dx.doi.org/10.1128/JCM.42.7.3137-3141.2004] [PMID: 15243072] , 15Eloff JN. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 1998; 64(8): 711-3.
[http://dx.doi.org/10.1055/s-2006-957563] [PMID: 9933989] , 22Espinel-Ingroff A, Barchiesi F, Cuenca-Estrella M, et al. International and multicenter comparison of EUCAST and CLSI M27-A2 broth microdilution methods for testing susceptibilities of Candida spp. to fluconazole, itraconazole, posaconazole, and voriconazole. J Clin Microbiol 2005; 43(8): 3884-9.
[http://dx.doi.org/10.1128/JCM.43.8.3884-3889.2005] [PMID: 16081926] , 23Khosravi AR, Riazipour M, Shokri H, Mousavi ML, Mahmoudi M. Characterization of the similarity of protein patterns and virulence of clinical Candida albicans isolates. J Biol Sci 2008; 8: 760-6.
[http://dx.doi.org/10.3923/jbs.2008.760.766] ] Comparative evaluation of 24-h visualization and 48-h spectrophotometric MIC end points to 48-h microdilution broth visual MICs of fluconazole, itraconazole, voriconazole, and posaconazole in Candida spp. reported that both the MIC endpoints results were similar. Moreover it was stated that spectrophotometric MICs are more effective than visual MICs [22Espinel-Ingroff A, Barchiesi F, Cuenca-Estrella M, et al. International and multicenter comparison of EUCAST and CLSI M27-A2 broth microdilution methods for testing susceptibilities of Candida spp. to fluconazole, itraconazole, posaconazole, and voriconazole. J Clin Microbiol 2005; 43(8): 3884-9.
[http://dx.doi.org/10.1128/JCM.43.8.3884-3889.2005] [PMID: 16081926] ]. This study carries significance duce to an increase in the antifungal resistance of Candida which is a lethal threat to immune compromised and hospital acquired infections. Intrinsic resistance to antifungal therapy had been reported . on various Candida species towards commonly used antifungal agents. It was also observed that the antifungal resistances are developed due to the treatment. It is very important to understand the mechanisms of drug resistance to improve the efficiency of treatment since Candida infections have high impact on immune compromised patients.

Table 2
Antifungal susceptibility and minimum inhibitory concentration of antifungal agent.

Out of 200 clinical isolates from 5 routes of infection (blood, catheter tip, high vaginal swab, oral and urine), it was observed that oral and catheter tips are the major sites where C. albicans infection can occur. In the study, it was also observed that catheter tip strains have high antibiotic resistant strains when considering other regions; it can be due to the pathological conduction of the patients that mediates the infection of resistant strain and the development of drug resistance. Thus the study signifies that catheter can be a factor for the development of antibiotic resistance since the procedure is carried out mostly on patients under antimicrobial treatment or immune compromised. It was also observed that the Candida isolated from oral route has higher antibiotic resistance pattern than viginal followed by UTI which is the prominent candida infection site. In this study, C. albicans showed 100% susceptibility against ketoconazole and amphotericin-B at 1 µg/ml, whereas fluconazole and itraconazole resistance strains showed susceptibility upto16 to 64µg/ml, thus signifying an increase in resistance pattern. The study reveals higher antibiotic resistance in the clinical samples which proves the risk in C. albicans management program due to the risk of development of drug resistance.