Antachopoulos Charalampos, Meletiadis Joseph, Sein Tin, Roilides Emmanuel, Walsh Thomas J
Pediatric Oncology Branch, National Cancer Institute, 10 Center Drive, Bethesda, MD 20892, USA.
Antimicrob Agents Chemother. 2007 Mar;51(3):881-7. doi: 10.1128/AAC.01160-06. Epub 2006 Dec 4.
The minimum effective concentration (MEC) used to assess the in vitro antifungal activity of caspofungin against Aspergillus spp. is a qualitative endpoint requiring microscopic examination of hyphae. We therefore developed a tool for the quantitative assessment of caspofungin activity against Aspergillus spp. at clinically applicable concentrations. Susceptibility to caspofungin (0.008 to 8 microg/ml) was studied for 9 A. fumigatus, 8 A. flavus, and 12 A. terreus isolates based on the Clinical and Laboratory Standards Institute M38-A protocol. After 48 h of incubation, the MEC was defined microscopically, and metabolic activity assessed with a modified XTT assay, using 100 microg of the tetrazolium salt XTT/ml and 6.25 muM menadione. A significant reduction in metabolic activity was demonstrated at the MEC (0.25 to 0.5 microg/ml) for all Aspergillus spp. and was more pronounced for A. flavus (median metabolic activity, 25% of control) compared to A. fumigatus and A. terreus (median metabolism, 42 and 53%, respectively), allowing determination of MEC with the XTT assay (93 to 100% agreement with microscopic MEC). Fungal metabolism tended to reach the lowest levels (median, 17 to 38% of control) one to two dilutions higher than the MEC, at the minimum metabolic activity concentration (MMC). For 5 of 9 A. fumigatus isolates, 6 of 12 A. terreus isolates, and 1 of 8 A. flavus isolates, a paradoxical increase in metabolism was observed at concentrations greater than the MMC. Sigmoid (E(max)) or bell-shaped models described accurately (median R(2) = 0.97) the concentration-dependent metabolic changes in the absence or presence, respectively, of paradoxical response. Assessment of metabolic activity may provide useful quantitative endpoints for in vitro studies of caspofungin against Aspergillus spp.
用于评估卡泊芬净对曲霉属真菌体外抗真菌活性的最低有效浓度(MEC)是一个定性终点,需要对菌丝进行显微镜检查。因此,我们开发了一种工具,用于在临床适用浓度下定量评估卡泊芬净对曲霉属真菌的活性。根据临床和实验室标准协会M38-A方案,研究了9株烟曲霉、8株黄曲霉和12株土曲霉分离株对卡泊芬净(0.008至8微克/毫升)的敏感性。孵育48小时后,通过显微镜确定MEC,并使用改良的XTT法评估代谢活性,该方法使用100微克四氮唑盐XTT/毫升和6.25微摩尔甲萘醌。所有曲霉属真菌在MEC(0.25至0.5微克/毫升)时均显示代谢活性显著降低,黄曲霉(代谢活性中位数为对照的25%)比烟曲霉和土曲霉(代谢活性中位数分别为42%和53%)更明显,这使得可以通过XTT法确定MEC(与显微镜下MEC的一致性为93%至100%)。在最低代谢活性浓度(MMC)下,真菌代谢往往在比MEC高1至2倍稀释度时达到最低水平(中位数为对照的17%至38%)。在9株烟曲霉分离株中的5株、12株土曲霉分离株中的6株和8株黄曲霉分离株中的1株中,在浓度高于MMC时观察到代谢出现反常增加。Sigmoid(E(max))或钟形模型分别准确描述了(中位数R(2)=0.97)在无反常反应或有反常反应情况下浓度依赖性的代谢变化。代谢活性评估可为卡泊芬净对曲霉属真菌的体外研究提供有用的定量终点。