Comparison of Results Obtained by Testing with Three Different Agar Media and by the NCCLS M27-A Method for In Vitro Testing of Fluconazole against Candida spp

Abstract

Fluconazole susceptibilities of 150 Candida isolates were determined by a 25-g fluconazole disk diffusion agar test and compared with the microdilution NCCLS M27-A method. The agar test used three different media and was read at 24 and 48 h. When only the susceptible and nonsusceptible categories were used, disk diffusion with Müeller-Hinton agar supplemented with 2% glucose and 0.5 g of methylene blue (MHGM) per ml had a 95.37% correlation with the MIC method at 24 h, followed by RPMI 1640-2% of glucose agar (correlation, 94%) and Shadomy medium (SHDM) (correlation, 92.6%). The growth of microcolonies inside the inhibition zones was common (>63%) in the RPMI and SHDM media and minimal with MHGM (8.7%). At 48 h, MHGM and SHDM still had a >91% correlation with the MIC, while RPMI results had dropped to 75%. The best overall agreement was obtained with C. dubliniensis (100%).Fungal infection with Candida species is still an important cause of morbidity and mortality in immunocompromised patients. With the widespread use of fluconazole for the treatment and prevention of oropharyngeal and/or esophageal candidiasis, particularly in AIDS patients, clinical resistance is becoming a serious problem (5,7,8,12,21).What is needed is a rapid, easy, reproducible, and inexpensive in vitro method of obtaining susceptibility data which can guide the treatment of clinical yeast infections.The M27-A and M27-A2 reference NCCLS methods for antifungal susceptibility testing (15, 17) are cumbersome and costly, and reading the endpoints of the azoles is difficult. Alternative methods such as the broth microdilution adaptation of the M-27A and M27-A2 methods or the E test are simplified tests but are not easily adapted to the screening of yeasts for fluconazole susceptibility, nor are they cost-effective enough to be performed routinely in most clinical microbiology laboratories (6,18,20,24).Recent studies (2, 4, 9, 11, 13, 14, 19; C. Durussel, A. M. Daoui-Hassani, and J. Bille, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. J120, 1998) have demonstrated that an agar disk diffusion method similar to that used for testing antibacterial agents (NCCLS M2-A6) (16) can reproducibly and accurately determine the susceptibility of yeasts to fluconazole and can easily be incorporated into a clinical laboratory as an effective means for fluconazole susceptibility screening.The aim of this report is to study the agreement between the broth microdilution NCCLS method and a 25-g fluconazole disk diffusion test performed using RPMI 1640-2% glucose agar (RPMIG), Müeller-Hinton agar supplemented with 2% glucose and 0.5 g of methylene blue (MHGM) per ml, and Shadomy medium (SHDM).A total of 150 isolates of Candida spp. were collected from the clinical microbiology laboratory of Hospital Clínico Universitario Lozano Blesa (Zaragoza, Spain) and submitted for fluconazole susceptibility testing by microdilution and agar diffusion. The Candida species tested were 63 C. albicans isolates, 25 C. dubliniensis isolates, 25 C. glabrata isolat...

Evaluation of glucose-methylene-blue-mueller-hinton agar for E-test minimum inhibitory concentration determination in <i>Candida </i>spp.

“…Despite being easy and practical, this needs to be conÞ rmed by BMD to exclude false resistance. 1 Recent reports have documented comparable results between BMD (NCCLS-27-A) 3 and agar-based E-test.…”

“…2,6 A large number of recent studies have compared E-test or DD on different media. 1,2,6 But studies comparing E-test MIC on RPMIG and GMB-MHA could not be seen in the pertinent literature. The overall levels of agreement between E-strip MICs obtained by RPMIG and GMB-MHA at 24 h were comparable (97% agreement) for both amphotericin-B and ß uconazole for resistant and susceptible isolates.…”

“…5 It was observed in the current and prior studies that GMB-MHA has enhanced growth, simpliÞ ed reading and minimal microcolonies around zone of inhibition. 1,4 Thus GMB-MHA appears to be a useful medium for E-test, as it compared well with BMD and RPMI-G (except a single C. tropicalis outlier despite repeat testing). The stock-solution of GMB can be refrigerated and added to the molten and cooled MHA which is made routinely.…”

“…The isolates included in the study comprised of C. tropicalis (12), C. parapsilosis (8), C. albicans (8), C. krusei (2) and C. glabrata (1). Twenty-four (77.4%) of the isolates that were found to be susceptible by BMD were identiÞ ed as susceptible by RPMI-G agar to amphotericin B and ß uconazole.…”

“…A rise in invasive fungal infections and their emerging resistance have necessitated the need for antifungal susceptibility testing (AFT) for clinical work-up. 1 The standardized broth micro-dilution (BMD) method is expensive, laborious and cumbersome for routine use in clinical microbiology laboratory. 2 Recently, a disc-diffusion method has been approved by CLSI using glucose-methylene-blue (GMB) Mueller-Hinton agar (MHA).…”