Quantitation of ergosterol content:: Novel method for determination of fluconazole susceptibility of Candida albicans
Quantitation of ergosterol content:: Novel method for determination of fluconazole susceptibility of Candida albicans
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DOI：
10.1128/jcm.37.10.3332-3337.1999
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发表时间：
1999-10-01
影响因子：
9.4
通讯作者：
Morrison, CJ
中科院分区：
文献类型：
--
作者：
Arthington-Skaggs, BA;Jradi, H;Morrison, CJ
最常用的唑类抗真菌药物氟康唑的MIC终点很难确定，因为在国家临床实验室标准委员会肉汤宏观稀释法和微量稀释法进行药敏测试时，其抑菌特性可能会导致过度的生长滞后。为了克服这种不确定性，以及由于氟康唑通过抑制麦角甾醇的生物合成而起作用，我们开发了一种新的方法来区分氟康唑敏感菌株和氟康唑耐药菌株，方法是定量：在0、1、4、16或单位浓度的氟康唑中生长的细胞产生麦角甾醇，通过皂化从整个酵母细胞中分离麦角甾醇，然后用庚烷提取非皂化脂质，麦角甾醇通过240-300 nm之间独特的分光光度吸光度曲线来鉴定。我们使用这种甾醇定量方法(Scm)对38株肉汤微量稀释点小于或等于8mU/ml(敏感)的菌株进行了检测。16~32mU g/ml(敏感的剂量依赖关系)，或大于或等于mU g/ml(耐药)和10株有拖尾终点的菌株的微量肉汤稀释法。在没有氟康唑的情况下生长的任何菌株之间的平均麦角甾醇含量没有显著差异。18株敏感株在1 mU g/ml氟康唑作用下麦角甾醇含量平均下降72%，4 mU g/ml作用后麦角甾醇含量下降84%，16 mU/ml和作用下麦角甾醇含量分别下降95%和100%。10株SDD菌株暴露于1、4、16和mU/ml氟康唑后，麦角甾醇平均下降38%、57%、73%和99%。相比之下，10株耐药菌株在暴露于相同浓度的氟康唑后，麦角甾醇含量仅平均下降了25%、38%、53%和84%。用SQM法计算氟康唑的最低抑菌浓度，即与未用药对照相比，氟康唑对麦角甾醇生物合成的抑制率达到80%或更高。在肉汤微量稀释法有清晰终点的38株菌株中，有33株(87%)的SQM MIC在肉汤微量稀释法的2倍稀释度以内。SQM还可区分耐药和高度耐药菌株，特别适用于另外10株因生长缓慢而通过肉汤微量稀释法给出可疑终点的菌株对氟康唑的敏感性。与肉汤微量稀释法相比，SQM测定的拖尾菌株是敏感的，而不是耐药的，这表明SQM可以更准确地预测临床结果，SQM可能为改进现有的氟康唑药敏试验方法提供了一种手段，并可能提供更好的体外和体内结果的相关性，特别是对于具有拖尾终点的菌株。
MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive "trailing" of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate 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 heptane, Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm, We used this sterol quantitation method (SQM) 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-dependent [SDD]), or greater than or equal to 64 mu g/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates 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, and 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, 57, 73, and 99% after exposure to 1, 4, 16, and 64 mu g of fluconazole/ml, respectively. In contrast, 10 resistant Isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in 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 resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal 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 may predict clinical outcome more accurately, The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.