Quantitation of ergosterol content: Novel method for determination of fluconazole susceptibility of Candida albicans

MIC end points for the most commonly prescribed azole antifungal drug, fluc onazole, can be difficult to determine because its fungistatic nature can l ead to excessive "trailing" of growth during susceptibility testing by Nati onal Committee for Clinical Laboratory Standards broth macrodilution and mi crodilution methods. To overcome this ambiguity, and because fluconazole ac ts by inhibiting ergosterol biosynthesis, we developed a novel method to di fferentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating: ergosterol production in cells grown in 0, 1, 4, 16, or 64 mu g of fluconazole per mi, Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with hept ane, Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm, We used this sterol quantitation method (SQ M) to test 38 isolates with broth microdilution end points of less than or equal to 8 mu g/ml (susceptible), 16 to 32 mu g/ml (susceptible dose-depend ent [SDD]), or greater than or equal to 64 mu g/ml (resistant) and 10 isola tes with trailing end points by the broth microdilution method. No signific ant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isol ates showed a mean reduction in ergosterol content of 72% after exposure to 1 mu g of fluconazole/ml, an 84% reduction after exposure to 4 mu g/ml, an d 95 and 100% reductions after exposure to 16 and 64 mu g of fluconazole/ml , respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 5 7, 73, and 99% after exposure to 1, 4, 16, and 64 mu g of fluconazole/ml, r espectively. In contrast, 10 resistant Isolates showed mean reductions in e rgosterol content of only 25, 38, 53, and 84% after exposure to the same co ncentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or gr eater inhibition of overall mean ergosterol biosynthesis compared to that i n the drug-free control. Of 38 isolates,which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resista nt and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equi vocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM m ay predict clinical outcome more accurately, The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may prov ide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.