Comparison between the Broth Microdilution and Disc Diffusion Techniques in Testing the Susceptibility of Candida Albicans to Fluconazole

Abstract

Antifungal drug susceptibility testing has become more important due to the increase in serious fungal infections and the concomitant emergence of resistance to antifungal agents. The reference methods for antifungal susceptibility testing are cumbersome, costly, and reading the endpoints of the azoles [as fluconazole] is difficult. Rapid, easy, reproducible, and inexpensive alternative methods of obtaining susceptibility data are needed. Hence, this study aimed at determination of the susceptibility patterns of clinical isolates of Candida albicans [C.albicans] to fluconazole [FLC] and comparison between the reference broth microdilution and disk diffusion methods [using two different agar media] for antifungal susceptibility testing. The study was carried out on 70 clinical C. albicans isolates [from 60 different cutaneous lesions in otherwise healthy patients and 100 immunocompromied patients complaining from symptoms of urinary tract infections]. Antifungal susceptibility testing of isolates was performed by the reference broth microdilution method and by the disk diffusion method on Yeast extract peptone dextrose [YEPD] agar & Mueller-Hinton agar supplemented with 2% glucose and 0.5 ug/ml of methylene blue [MHGM] using 25-ug FLC disk. The results revealed that, by the reference method 95.8% were susceptible [S], 1.4% was susceptible-dose dependent [S-DD], and 2.8% were resistant [R]. All the cutaneous isolates were susceptible to FLC compared to only 88% of the urinary ones. Comparison between MIC categories of the broth method and 24-h disk test categories on MHGM showed that, the observed agreement was 98.6%. When S-DD &R were considered as one category [non susceptible], the observed agreement increased to reach 100 %[ perfect agreement, k=1, p=0.000]. After 48-h incubation the observed agreement declined to 97.1%. When susceptible and non susceptible categories were considered, the observed agreement reached 98.6% [excellent agreement] [k=0.85,p=0.000]. On YEPD agar, the observed agreement was 97.1% [good agreement, k=0.74]. After 48-h, the observed agreement declined to 92.9% [fair agreement, k=0.51, p=0.000]. The inhibition zones had clear and definite margins in 97.1% of isolates on MHGM compared to 85.7% on YEPD. In conclusion, FLC has perfect antifungal effect on C.albicans isolated from cutaneous lesions in otherwise healthy patients while some of its activity is lost in isolates from the immunocompromised patients. The 25-ug fluconazole disk diffusion technique using MHGM agar interpreted after 24h correlated well with the reference microbroth method. Moreover, it is rapid, simple, convenient, cost effective, less subjective and less cumbersome than the reference method. Hence, FLC should be prescribed with caution and on strict indications to reduce the potential development of resistance. The use of FLC disk diffusion susceptibility testing of C.albicans on MHGM agar read after 24h could be recommended as an alternative to the reference micro- dilution method in routine practice.