Prevalence, Antifungal Susceptibility and Biofilm Characterization of Candida Species Isolated from Tertiary Care Hospitals, Rawalpindi, Pakistan

Abstract

It is becoming progressively more understandable that during the last few decades, development in modern health facilities has resulted in an increase in the incidence of fungal infections, globally. The leading cause of these fungal infections is the Candida species which is part of normal flora of 80% of the healthy individuals. Candidiasis (infections caused by Candida) has become a serious health problem owing to its high rate of morbidity and mortality. In Pakistan, despite of its high mortality and morbidity rate, little attention is devoted towards its study and control because of the limited lab resources. The present study was designed to investigate the prevalence, antifungal susceptibility and biofilm characterization of the Candida species. For this purpose, clinical isolates of Candida species were collected from 8 wards of 2 tertiary care hospitals during the period January 2011 to December 2012. The isolate(s) were identified on the basis of colony morphology, gram staining, germ tube test, growth on chromagar and corn meal agar and sugar assimilation profile. The antifungal susceptibility tests were done by disc diffusion and broth microdilution tests and results were interpreted by CLSI-criteria. Biofilms were formed on serum treated silicone elastomer discs and characterized by dry weight determination and XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay. The morphology of the biofilms was studied by fluorescence and confocal scanning laser microscopy. The antifungal susceptibility test of Candida biofilms was done by microtitre plate method by using XTT assay. We isolated 456 clinical isolates of Candida from various types of specimen. The germ tube test differentiated Candida albicans from non-albicans Candida with 98.4% sensitivity and 96.2 % specificity, respectively. On the basis of Chromagar and API (analytical profile index) test results, all the isolates were characterized into five species of Candida i.e., Candida albicans, Candida tropicalis, Candida glabrata, Candida kruseii and Candida parapsilosis. Candida infection was more common in females (63.4%) as compared to male patients (36.6%). Candida was most abundantly isolated from urine (35.5%), followed by high vaginal swabs (29%), sputum (11.4 %), pus (7.7 %), Central Venous Catheters (CVC) (9.4 %), wounds (4%), body fluids (2.2%), ear swab (0.4%), stool samples (0.4%), nasal swab (0.2%), nail clippings (0.2%). The maximum number of samples was obtained from the group of 21-40 years patients. Half of the isolated samples were received from the Medical Unit (33%) and Gynecology Ward (28%). Candida species were isolated from Medical Intensive Care Page x Unit (15%), Surgical Unit (7%), Pediatric Ward (6%), Outdoor Patient Department (6%), Urology Ward (3%) and Burn Unit (2%). It was seen that Candida albicans [56.1% (n=256)] was dominant over the non-albican species of Candida [43.9% (n=200)]. The most common species after Candida albicans was C. tropicalis (26.5%), followed by Candida glabrata (11.6%), Candida kruseii (3.7%), and Candida parapsilosis (2%). About 91% (n=413) of the Candida species were susceptible to all the 04 antifungals drugs tested against them. Their susceptibility against amphotericin, fluconazole, voriconazole and caspofungin was 99.6%, 92.5%, 98.1%, 96.7%, respectively. The resistance to antifungal drugs was significantly more common in non-albicans Candida [18.5% (37/200)] as compared to Candida albicans [2.3% (6/256)]. Candida albicans was the most susceptible species with only 2.3% resistance followed by C. tropicalis (2.7%), C. parapsilosis (11.1%), C. glabrata (20.8%) and C. kruseii (100%). The comparison of both antifungal susceptibility testing methods showed that highest agreement between the methods was found for caspofungin (97.8%) followed by amphotericin (96.4%), voriconazole (93.6 %) and then fluconazole (91.2%). Cross-resistance against fluconazole and voriconazole was also seen in almost every specie of Candida. It was found that Candida species formed highly resistant biofilms. Candida albicans was found to be the more biofilm producer with higher XTT activities and D followed by C. parapsilosis, C. tropicalis and C. glabrata. Candida glabrata had lesser ability to form biofilm as compared to other species. It was seen that the Candida strains isolated from CVC and HVS produce more biofilms as compared to the strains isolated from urine. However, all of these biofilms exhibited very high resistance to fluconazole. Caspofungin was found to be effective against the biofilms formed by Candida species. In contrast, caspofungin demonstrated low MIC values, showing activity against the biofilms. However, some strains of Candida glabrata exhibited high MIC against caspofungin in planktonic form and likewise exhibited increased resistance in biofilm form. There is a need that such studies should be conducted from time to time so as to monitor the trends of resistance against antifungal agents. This study should be extended to other hospitals comprising a large sample size and more antifungal agents should be tested against Candida species. The results confirmed high drug resistance in Candida biofilm that paves way for studies of other pathogenic factors such as protrease and phospholipase along with their relationship to the biofilm formation. It would be of immense value to establish candidiasis surveillance program in order to monitor changes in incidence and resistance rate.