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Abstract：
A common strategy in antifungal susceptibility testing is the utilization of the standardized protocol based on the microbroth dilution assay approach as described by the Clinical Laboratory Standards Institute (CLSI) (M27-A4). One major problem for laboratories in resource-limited countries with this protocol arises from the use of expensive culture media like RPMI-1640 and 3-N-morpholinopropanesulfonic acid (MOPS) buffer. One approach of circumventing this problem in cases of economic need is the evaluation of alternative culture media and buffers. The overall goal of this work was to investigate the influence of modifications in the protocol M27-A4 on diagnostic reliability. We performed univariate analyses evaluating (1) 2 different culture media (YNB and modified SAB); (2) three different buffers (sodium bicarbonate, Tris-HCL, and phosphate), as well as the influence of inoculum concentration (102, 103, 104, 105 cells/mL), the influence of incubation time, and the influence of the assessment mode (visual, biological dye, and spectrophotometer). Our results suggested that (1) RPMI-1640 may be substituted by modified SAB and (2) MOPS buffer may be substituted by Tris-HCl buffer for defined analyses. By comparing the CLSI protocol and the alternative protocol proposed in the present study (modified SAB and Tris-HCl buffer) for the assessment of fluconazole susceptibility of eighteen yeasts (clinical isolates), similar results with both methodologies were recorded. We feel that this study should stimulate a discussion on the feasibility and evolution of the M27-A4 protocol in order to include pragmatic alternatives for resource-limited settings.
摘要：
抗真菌药敏试验中的常见策略是利用基于临床实验室标准研究所（CLSI）（M27-A4）所述的微量肉汤稀释测定方法的标准化方案。对于资源有限国家的实验室而言，该方案的一个主要问题是使用昂贵的培养基，例如RPMI-1640和3-N-吗啉代丙磺酸（MOPS）缓冲液。在经济需要的情况下规避该问题的一种方法是评估替代培养基和缓冲液。这项工作的总体目标是研究方案M27-A4中的修改对诊断可靠性的影响。我们进行了单变量分析，评估（1）2种不同的培养基（YNB和改良的SAB）；（2）三种不同的缓冲液（碳酸氢钠，Tris-HCL,和磷酸盐)，以及接种浓度的影响（102，103，104，105细胞/mL），孵化时间的影响，以及评估模式的影响(视觉，生物染料，和分光光度计）。我们的结果表明，（1）RPMI-1640可以用修饰的SAB代替，（2）MOPS缓冲液可以用Tris-HCl缓冲液代替，用于确定的分析。通过比较CLSI方案和本研究中提出的替代方案（改良的SAB和Tris-HCl缓冲液），用于评估十八个酵母（临床分离株）的氟康唑敏感性，两种方法的结果相似.我们认为，这项研究应激发对M27-A4协议的可行性和演变的讨论，以便包括针对资源有限环境的实用替代方案。
