Antifungal Susceptibility Pattern of Candida Albicans in Human Infections

Results: All isolates in the study were sensitive to amphotericin-B and ketoconazole and a high frequency of fluconazole and itraconazole resistance was observed. Oral and catheter tips were observed to be the major sites of C. albicans infections. Significant resistance aganist fluconazole (56.5%) and itraconazole (64.5%) was observed with MIC at 16, 32 to 64μg/ml. All isolates were observed to be sensitive for ketoconazole and amphotericin-B at 0.5μg/ml and 1μg/ml.


INTRODUCTION
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 [1,2].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 [3].About 75% women have this fungus without it causing infection [4,5].The factors that predispose women to vaginal candidiasis are changes in pH, use of oral contraceptives, tight clothing, and personal hygiene [6,7].
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 [8].Azole-resistant C. albicans is frequently observed in oropharyngeal candidiasis of HIV patients [9].Apart from albicans, species of Candida like C. glabrata, C. krusei, and C. lusitaniae have been reported with reduced its susceptibility to fluconazole [10,11].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 [13].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.

Sample Collection
Pathological strains of C. albicans from five different sources viz., blood (B), catheter tip (CT), high vaginal swab (HVS), oral (O) and urine (U) were collected from the Department of Microbiology, People's College of Medical Sciences and Research centre(PCMS&RC) and Peoples Dental Academy, People's University, Bhopal.The culturing of C. albicans from CT, HVS, O and U and was done on Sabouraud Dextrose Agar (SDA) medium and primarily B samples on blood culturing media and transfer to SDA.The strains were confirmed based on its culture characteristics and microscopic observation.

Antifungal Susceptibility
Well diffusion antifungal susceptibility assay was carry out by the method proposed by Magaldi [14].SDA plates with inoculums were assayed in the wells made using sterilized cork borer, to which, antifungal agents amphotericin B (100 U), ketoconazole (20 μg/mL), itraconazole (20μg/mL), and fluconazole (20 μg/mL) were loaded.The plates were incubated at 37 • for 48 hours and the zone of inhibition was recorded.

Minimum Inhibitory Concentration
Using microdilution assay, the minimal inhibitory concentration (MIC) against fluconazole, itraconazole, ketoconazole and amphotericin-B was determined based on the method of Eloff [15].96 well plates with 100 μl of C. albicans inoculums in sabouraud dextrose broth were cultured for 48 hours at 37˚C.The stock solution (100µl/ml) of antifungal agents amphotericin B, ketoconazole, itraconazole and fluconazole was serially diluted (0.5 to 64µg/ml) and added to separate wells along with 100µ/ml sabouraud dextrose broth.The plates were incubated at 37˚C for 48 hours and the MIC value was determined by reading the intensity of the colour produced in the wells after the addition of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), defining the cell concentration in the well.

RESULT AND DISCUSSION
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 [16].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. [17], 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 [18] where decreased susceptibility to azoles in C. albicans.was observed.It is also reported similar susceptibility of C. albicans [19].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 [20,21].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 [12,15,22,23] 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 [22].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.

CONCLUSION
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.