Influência de modificações na técnica de microdiluição em caldo (CLSI, protocolo M27-A3) na determinação de sensibilidade de leveduras ao fluconazol

Abstract

One of the main criticisms of the Clinical Laboratory Standards Institute (CLSI) standard protocol for studying antifungal activity - protocol M27-A3 is the cost of the RPMI-1640 growth medium and the MOPS (3- (N-morpholino) propanesulfonic acid) buffer. Due to these limitations, the present study aimed to investigate the influence of modifications in the broth microdilution technique (CLSI, protocol M27-A3) on the determination of fluconazole yeast sensitivity. To evaluate this objective univariate assays were performed modifying the test recommended by CLSI protocol M27-A3. Yeasts used in the trials belonged to Candida and Cryptococcus genera. The modifications made to the M27-A3 protocol were: a) culture media change (five media were evaluated: YNB medium and four different Sabouraud composition variations); b) buffer type (sodium bicarbonate, tris-HCL and phosphate), inoculum concentration (102, 104, 105 cells / mL), incubation time (24 and 48 h) and reading form (visual, dye and spectrophotometer) ) in the MIC of yeast in front of fluconazole. The different culture media evaluated (except 2% dextrose medium and 1% casein peptone) resulted in similar MIC results to assays performed with standard RPMI medium. Tris-HCl buffer assays resulted in similar MIC results to those performed with standard MOPS buffer. The concentration of 1x10 3 cells / mL was the only concentration tested that resulted in results similar to the standard protocol. Only the incubation time of the 24hs trial proved to be suitable for the Candida genus. Direct visual reading, visual dye use (resazurin) and spectrophotometry showed similar results. From the knowledge obtained in the univariate experiments a new protocol was proposed: a) culture medium: dextrose 5 g / L and soybean peptone 10 g / L; b) buffer: Tris-HCL; c) 0.5-2.5 x103 inoculum, d) 24 hr assay for Candida spp and 48h for Cryptococcus neoformans and e) direct visual reading. The analysis of variance showed 95% regression comparing the MIC results of 19 yeasts submitted to antifungigram by CLSI protocol and the one proposed in the present study. These results are important and robust and present a more affordable option for antifungigram assays. However, a multicenter study with a larger number of microorganisms is needed to evaluate the effect of the medium on intra and interlaboratory variations in fluconazole MICs for yeast.