The present prospective observational study was conducted in the Department of Microbiology in association with Department of Pharmacy during one year period from June 2011-July 2012. A total of 1376 patients of all age groups and sex visiting the OPD and admitted to the hospital with clinical suspicion of fungal septicaemia were included in the study. Patients who were already on antifungals were excluded from the study. Demographic and clinical data such as age, sex, birth weight, history of pre-term birth, antibiotic prophylaxis history, presence of any co-morbid condition and presence of any intravenous lines were noted for all these patients. Consecutive blood samples in duplicate were collected from each patient under aseptic precautions in two sets of biphasic Brain Heart Infusion (BHI) medium. One BHI set was incubated at 25°C and other at 37°C and subcultures were performed after 24 hours, 48 hours and 72 hours up to 7 days. The colonies were identified by standard microbiological techniques. A total of 100 Candida isolates were obtained and identified up-to the species level on the basis of germ tube test, morphology on cornmeal agar, growth on Hi-Chrome Candida agar, carbohydrate fermentation and sugar assimilation test [5,6].

The isolated Candida isolates were further subjected to antifungal susceptibility testing against fluconazole (Hi-media), voriconazole (Pfizer), cinnamon oil and olive oil. Susceptibility testing for fluconazole and voriconazole was performed by the broth microdilution minimum inhibitory concentration (BMD-MIC) method using RPMI (Roswell Park Memorial Institute) medium and MOPS (3-(N-morpholino) propane sulphonic acid) buffer. MIC results were interpreted as per NCCL (M-27-A2) guidelines. The quality control test was performed using ATCC strain, C. albicans ATCC 90028, C. parapsilosis ATCC 22019, Candida tropicalisATCC750 and Candida krusei ATCC 6258 [7]. All the 100 isolates were subjected to susceptibility testing against cinnamon oil and olive oil by agar well diffusion method. Candida strain to be tested was inoculated on sterilized Sabouraud’s Dextrose Agar plate. A hole was punched aseptically by sterile cork borer of 6-mm diameter on the agar surface. A 50 μl of cinnamon and olive oil was introduced into each of the peripheral wells with sterilized DMSO as negative control. The plates were incubated at 28°C. Antimicrobial activity of essential oils was analysed by observing the zone of inhibition. The results were interpreted as <9 mm- inactive; 9-12 mm- partially active; 13-18 mm- active; >18 mm- very active [8,9]. Statistical analysis was performed by chi-square test using SPSS version- 16.0 software.

During the study period, a total of 100 (7.26%) isolates of Candida spp. were isolated from 1376 patients. Rate of Candida isolation was higher in paediatric (1-15years) age group (89%) as compared to adults (> 15 years) (11%).

Majority of Candida strains were isolated from neonates (36%) followed by infants (28%), 1-15 years (25%) and adults (11%) [Table/Fig-1]. The male to female ratio was 3:1. The rate of Candida isolation was maximum from PICU (Paediatric Intensive Care Unit) patients (72%) followed by wards (21%) and OPD’s (7%) [Table/Fig-2].

Out of the 100 Candida isolates, C. tropicalis (43%) was the most common isolate followed by C. albicans (41%), C. krusei (9%) and C. parapsilosis (7%). C. albicans was more frequently isolated from the infants (0-1month) whereas C. tropicalis was more common in the neonates (1-12 months) [Table/Fig-1]. All species were predominant in males than females. The most common risk factor among paediatric age group was I/V line (92%) followed by premature birth (73.03%), low birth weight (65.17%) and use of prior antibiotics (60.67%) [Table/Fig-3,4]. In adults, the most common risk factor was central venous line (90.9%), followed by ICU admission (63.64%), prior use of antibiotics (45.4%), patients on ventilator (36.4%), and immune-compromised status (18.18%). Antifungal susceptibility results revealed that C. albicans (85.3%) and C. parapsilosis (85.7%) were found to be most sensitive to fluconazole followed by C. tropicalis (67.4%). All Candida isolates were 100%sensitive to voriconazole. For olive oil, sensitivity of C. albicans was 53.65%, C. tropicalis was 48.83%, C. kruseiwas 55.55% and C. parapsilosis was 57.12%. Sensitivity to Cinnamon oil was 58.83% for C. albicans, 46.51% for C. tropicalis, 44.45% for C. krusei and 57.12% for C. parapsilosis. The resistance to fluconazole as well as olive oil and cinnamon oil was significantly higher (p-value > 0.05) in non albicansCandida spp. as compared to C. albicans [Table/Fig-5,6 and 7].

[Table/Fig-4]:

Predisposing risk factors among pediatric patients.

[Table/Fig-6]:

Resistance pattern of various condida isolates against antifungal drugs and oils.

Candidemia has emerged as an alarming problem in healthcare settings since last few decades causing increased morbidity and mortality [10]. In our study, the rate of isolation of Candida spp. from blood stream infections was 7.25%. Our study co-relates with previous studies conducted in India and Thailand, which reported rate of Candidemia as 6.9% and 6.14% respectively [11,12]. In contrast to our study, few studies from Pennsylvania and Saudi Arabia showed isolation rates as low as 3.5% and 2.8% respectively [13,14]. The difference in isolation rates in these studies could be due to the difference in antibiotic policies in different hospitals, as injudicious use of antibiotics leads to suppression of normal flora making the patients prone to develop Candidemia. The various risk factors for acquisition of Candidemia can be ICU settings, extremes of age and previous antibiotic therapy [15]. Our study showed a higher rate of Candidemia (72%) in ICU settings as well as in neonates (36%). Various studies across the globe have also observed a higher incidence of Candidemia in ICUs as compared to non ICU locations [1,15]. Ours is a tertiary care hospital and higher rate of Candidemia in our ICU can be explained by the fact that most patients were already on antibiotics, moreover about 48% of our study population consisted of neonates who generally have a suppressed immune system. Higher rates of Candidemia in neonates have been documented by various studies done earlier also [16,17]. In our study, the most common species was C. tropicalis followed by C. albicans, C. krusei and C. parapsilosis co-inciding with the observations from various other studies, which also depict a shifting trend from albicans to non-albicansCandida spp [1,18,19]. The rate of fluconazole resistance in this study is higher than the previous study done at our center as well as at some other parts of North India [20,21]. There is growing evidence that increasing use of azole agents may be associated with increasing fluconazole resistance as well as emergence of non-albicansCandida spp [22]. The high rate of resistance in the present study may be due to the irrational and liberal use of fluconazole or acquisition of resistance by previously susceptible strains of C. albicans following long term azole exposure. Some non- albicansCandida spp like C. krusei and C. glabrata exhibit resistance to traditional triazole antifungals like fluconazole. Voriconazole can be used effectively for the patients with infections due to C. kruesi and fluconazole resistant voriconazole susceptible C. glabrata, however, these spp may also demonstrate cross resistance to voriconazole resulting in treatment failure [23,24]. Such reports have encouraged looking for the alternative new approaches with low potential for development of resistance. We studied the efficacy of cinnamon oil and olive oil against Candida spp including C. krusei which is intrinsically resistant to fluconazole. Besides having antimicrobial properties, these compounds have other beneficial health effects including antioxidant and anti-inflammatory properties and most importantly free from any systemic side effects [25]. In our study although 50% of the isolates were resistant to both the oils but encouraging finding was that about 55.5% of C. krusei strains were sensitive to olive oil and cinnamon oil. Similar study, has also demonstrated that cinnamon oil and olive oil have marked antimicrobial activity against the fluconazole resistant C. albicans strain (ATCC-10231) [26]. In the present study all the C. krusei strains from Candidemia patients were sensitive to voriconazole but cross resistance may lead to voriconazole resistance resulting in treatment failure [23,24]. Thus these plant oils can find use as anti-Candida agents in future against azole resistant strains.

One of the potential limitations of the study is that we did not perform in vivo study. In vitro and in vivo results may not correspond with each other.

We conclude that fluconazole resistant non-albicansCandida has emerged as a major cause of Candidemia especially in neonates and ICU patients. Voriconazole still continues to be a promising drug at our center. Cinnamon oil and olive oil showed marked sensitivity against the fluconazole resistant C. krusei.