Rapid Identification and Antifungal Susceptibility Pattern of Candida Isolates from Critically Ill Patients with Candiduria

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1 Original Article Vol. 26 No. 2 Rapid identification and antifungal susceptibility pattern of Candida isolates:- Chaudhary U, et al. 49 Rapid Identification and Antifungal Susceptibility Pattern of Candida Isolates from Critically Ill Patients with Candiduria Uma Chaudhary, M.D.*, Antariksh Deep, M.D.**, Narender Chabbra, M.D.*** ABSTRACT Urinary tract infections are among the most common infectious diseases in humans. The incidence of candidiasis is on the rise in hospitalized patients especially due to non-albicans Candida. Early specific identification of the infected yeasts provides important help in selection the appropriate choice of antifungal agents since some Candida species for example Candida krusei is inherently resistant to fluconazole. The present study was aimed to isolate, identify, and perform antifungal susceptibility testing of the yeast isolates from the urine samples of patients hospitalized in the intensive care unit. The sensitivity of HiCrome Candida agar for identification of the species of Candida was also investigated, in comparison with standard conventional method. Candida albicans was the commonest species isolated, followed by C. dubliniensis, C. krusei, C. tropicalis, and C. glabrata. The sensitivity of HiCrome Candida agar for the identification of the species of Candida ranged from 88 percent to 0 percent. Amphotericin B was found to be the most effective antifungal agent, followed by itraconazole, ketoconazole, and fluconazole. The time taken for identification of the species of the yeasts by the conventional methods ranged from 96 to 120 hours, whereas it was only 48 hours with HiCrome Candida agar. (J Infect Dis Antimicrob Agents 2009;26:49-53.) INTRODUCTION Urinary tract infections are among the most common infectious diseases in humans, and large numbers of clinical samples are processed daily in most clinical laboratories. 1 The incidence of candidiasis is on the rise in hospitalized patients. Epidemiological surveillance indicates that Candida species are now the fourth most common pathogen causing blood-stream infections. 2 Also in the past decade, non-albicans Candida causing both blood-stream and urinary tract *Senior Professor & Head, Department of Microbiology, Pt. B.D. Sharma, PGIMS, Rohtak, India. **Assistant Professor, Department of Microbiology, Pt. B.D. Sharma, PGIMS, Rohtak, India. ***Post Graduate Student, Department of Microbiology, Pt. B.D. Sharma, PGIMS,Rohtak, India. Received for publication: April 2, Reprint request: Dr. Antariksh Deep, Assistant Professor, Department of Microbiology, Pt. B.D. Sharma, PGIMS, Rohtak drantarikshdeep@hotmail.com Keywords: Urinary tract infection, Candida, HiCrome Candida agar, susceptibility 49

2 50 J INFECT DIS ANTIMICROB AGENTS May-Aug infections are on the rise. In many cases, the newly recognized species are associated with candidemia, infected catheters, or onychomycosis. 3-5 Nosocomial candidal infections are important causes of morbidity and mortality in patients hospitalized to the intensive care units. 6 Urinary catheters have been held responsible for 80 percent of the hospital-acquired urinary tract infections. Surveillance data (1986 to 1996) from the United States National Nosocomial Infection Surveillance System has shown that Candida albicans is the fourth most common cause of urinary tract infections. 7 Conditions that increase the rates of genital Candida colonization are known risk factors for candiduria including diabetes mellitus, antibiotic use, immunosuppressive therapy, prolonged hospitalization, extremes of age, and female sex. 8- Recently, the incidence of life-threatening fungal infections has been the rise, and rapid identification of pathogenic yeasts and detection of polyfungal infections has become mandatory. The most widely used medium for the isolation of Candida species and other fungi is Sabouraud s dextrose agar. As the identification of yeasts may take several days, employing chromogenic media can help to reduce the time of isolation and identification as well as the detection of mixed cultures by hours. This can help clinicians in optimizing the selection of antifungal agents and provide a more rational and customized therapy. The present study was aimed to isolate, identify, and perform antifungal susceptibility testing of the yeast isolates from the urine samples of patients hospitalized in the intensive care unit and the predisposing factors associated with candiduria. The sensitivity of HiCrome Candida agar media (HiMedia Laboratories, Mumbai) for identification of the species of Candida was also investigated, in comparison with standard conventional method. PATIENTS AND METHODS The study was carried out in Department of Microbiology, Pt. BDS, University of Health Sciences, Rohtak, Haryana, India. Both catheterized and clean catch urine samples, collected on 2 or 3 different occasions from patients hospitalized in the intensive care units (ICUs) were subjected to routine microscopy. The isolated Candida was considered to be significant if the species of Candida was recovered from the urine of the patient on at least two consecutive days, and the colony count was > 5 colony-forming units/ml. The urine samples were spreaded by calibrated loop (0.01 ml) onto cystine lactose electrolyte deficient agar, Sabouraud s dextrose agar, and HiCrome Candida agar. Inoculated media were incubated at 37 C, and read after 24 and 48 hours of incubation. Yeast isolates were identified on routine microscopy, by growth pattern on Sabouraud s dextrose agar, cystine lactose electrolyte deficient agar, and the typical colony morphology on HiCrome Candida agar (HiMedia Laboratories, Mumbai). In addition, Candida albicans was identified by the germ tube formation method. C. albicans (ATCC 231, luxuriant light green growth), C. tropicalis (ATCC 750, luxuriant light blue growth), C. krusei (ATCC 24408, luxuriant white fuzzy growth), Eschererichia coli (ATCC 25922, inhibited) and Staphylococcus aureus (ATCC 25923, inhibited) were used as control strains for quality control of HiCrome Candida agar. The sugar fermentation and assimilation tests were taken as the gold standard for differentiation of Candida species. The carbohydrate assimilation test was carried out on yeast nitrogen base agar. Commercially available (HiMedia Laboratories, Mumbai) carbohydrate disks of 4-percent concentration including dextrose, maltose, sucrose, lactose, raffinose, mellibiose, xylose, and trehalose were used for performing the sugar assimilation tests.

3 Vol. 26 No. 2 Rapid identification and antifungal susceptibility pattern of Candida isolates:- Chaudhary U, et al. 51 Antifungal susceptibility testing was carried out using the disk diffusion method following the M 44-A National Committee for Clinical Laboratory Standards (NCCLS) guidelines, using fluconazole, itraconazole, ketoconazole, and amphotericin B antifungal disks. 11 Supplemented Mueller-Hinton agar [Mueller-Hinton agar + 2% glucose and 0.5 μg/ml methylene blue dye, (GMB medium)] was used for performing the antifungal susceptibility testing. surgical procedures (33%). The sensitivity of HiCrome Candida agar for the identification of the species of Candida ranged from 88 percent to 0 percent. Amphotericin B was found to be the most effective antifungal agent, followed by itraconazole, ketoconazole, and fluconazole (Table 2). The time taken for identification of the species by the conventional methods ranged from 96 to 120 hours, whereas it was only 48 hours with HiCrome Candida agar. RESULTS The urine samples from a total of 4,654 patients hospitalized in the ICUs over a period of two years were obtained. Candida species were isolated from 250 patients. Therefore, the prevalence candiduria in critically ill patients in our institute was 5 percent. Females were affected predominantly (73%). C. albicans was the commonest species isolated, followed by C. dubliniensis, C. krusei, C. tropicalis, and C. glabrata (Table 1). Antibiotic pressure was identified as the commonest (99%) predisposing factor, followed by the presence of indwelling devices (90%), prolonged hospitalization (85%), female sex (73%), and previous DISCUSSION A broad diversity of Candida species was observed in the present study. The prevalence of candiduria caused by the species other than C. albicans was surprisingly high (66%). In the present study, the most common species of Candida isolated from the patients admitted in the ICUs was C. albicans (34%), followed by C. dubliniensis (31%). In fact, nonalbicans Candida accounted for 66 percent of all Candida isolates. An early identification of the species of Candida provides an important help in selection appropriate antifungal treatment, since C. glabrata and C. krusei are usually resistant to fluconazole Table 1. The identification of Candida species based on biochemical tests performing on standard routine Sabouraud s dextrose agar (SDA) and HiCrome Candida agar. Species Conve ntional me thod (SDA) (N=250) HiCrome Morphology color Candida agar Se nsitivity C. albicans 86 (34) L ight green 84 (98) C. dubliniensis 78 (31) D ark green 74 (95) C. krusei 48 (19) W hite 47 (98) C. tropicalis 37 (15) B lue 32 (88) C. glabrata 1 (1) L ight pink 1 51

4 52 J INFECT DIS ANTIMICROB AGENTS May-Aug Table 2. Antifungal susceptibility pattern of isolates Candida species. Species Amphote ricin B Fluconazole Itraconazole Ke toconazole C. albicans (N=86) C. dubliniensis (N=78) C. krusei (N=48) C. tropicalis (N=37) C. glabrata (N=1) 86 7 (8) 20 (23) 12 (14) 78 5 (6) (13) 5 (6) 47 (98) 1 (2) (21) 3 (6) 37 (27) 19 (24) 9 (24) 1 In the present study, prior or current receipt of antibiotic therapy (99.6%), indwelling urinary catheter (90%), prolonged hospitalization (85%), female sex (73%), and surgical procedures (33%) were identified as the predominant risk factors for candiduria. The duration of urinary catheterization perhaps cannot be ignored as a risk factor. The time intervals of 6, 7, or 12 days have been reported by several studies for the colonization of Candida after the insertion of urinary catheters. 15,16 In the present study, the mean duration of urinary catheterization was 7 days. In the present study, antibiotic pressure was identified as the commonest risk factor for candiduria. It is known that suppressing susceptible endogenous bacterial flora in the gastrointestinal and lower genitourinary tracts, as a result of antibiotic therapy, results in fungal colonization of uroepithelial surface with ready access to the urinary tract, especially in the presence of indwelling urinary catheters. 17 In the present study, candiduria was found to be higher in female patients than in male patients, in consistent with other previous studies. 6 The higher prevalence in female patients may reflect vaginal candidiasis as the yeasts may ascend from the genitourinary tract to the urinary tract. This explanation was described by Febre and colleagues who in their study found that five of eight patients with positive vaginal secretions later showed the presence of the same species of Candida in their urine. 6 In conclusion, it can be realized that candiduria is a common problem in critically ill patients, and may get resolved spontaneously depending on whether the risk factors can be corrected. Most of the risk factors associated with candiduria in the present study can be controlled. The impact of faster identification of the species of Candida in patients with candidal urinary tract infection will help the clinician in selecting the appropriate antifungal agent, and thus contributing to overall reduction in the cost of treatment and the duration of hospital stay. References 1. Arao S, Matsuura S, Nonomura M, Miki K, Kabasawa K, Nakanishi H. Measurement of urinary lactoferrin as a marker of urinary tract infection. J Clin Microbiol 1999;37: Horvath LL, Hospenthal DR, Murray CK, Dooley DP. Direct isolation of Candida spp. from blood cultures

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