Division of Infectious Diseases and International Health, Duke University School of Medicine, Durham, NC, USA.
Trinity College of Arts and Sciences, Duke University, Durham, NC, USA.
Med Mycol. 2023 Nov 6;61(11). doi: 10.1093/mmy/myad111.
Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24 h post-inoculation and for MIC reading to be 72 h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008-0.025 μg/ml for itraconazole, 0.004-0.13 μg/ml for voriconazole, 0.03-0.13 μg/ml for posaconazole, 0.06-0.5 µg/ml for flucytosine, 0.5-1 µg/ml for amphotericin B, 0.5-4 µg/ml for caspofungin, and 0.5-16 µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%-100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.
抗真菌药物耐药性是侵袭性真菌感染治疗失败的一个新出现的原因，抗真菌药敏试验（AFST）可能为治疗决策提供信息。目前，尚无关于马尔尼菲青霉（Tm）或其他双相真菌的既定 AFST 指南。我们开发了一种使用荧光氧化还原指示剂 alamarBlue 的比色 AFST 方法，该指示剂的颜色会根据细胞代谢活性从蓝色变为粉红色。我们确定 alamarBlue 最佳添加时间为接种后 24 小时，MIC 读数时间为接种后 72 小时。我们的方法允许通过三种方式确定最小抑菌浓度（MIC）：颜色变化的目视检查、光密度和荧光强度。我们通过确定七种抗真菌药物对 32 株 Tm 临床分离株的 MIC 值来验证该测定法，并评估我们的 alamarBlue 与临床实验室标准化协会（CLSI）肉汤微量稀释方法之间的基本一致性（EA）和评分者间可靠性。MIC 范围（从低到高）为：伊曲康唑 0.008-0.025μg/ml，伏立康唑 0.004-0.13μg/ml，泊沙康唑 0.03-0.13μg/ml，氟胞嘧啶 0.06-0.5μg/ml，两性霉素 B 0.5-1μg/ml，卡泊芬净 0.5-4μg/ml，氟康唑 0.5-16μg/ml。alamarBlue 方法的所有三种 MIC 读数之间的 EA 均为 100%，alamarBlue 方法与 CLSI 方法之间的 EA 为 94%-100%。与既定的 CLSI 方法相比，我们的 alamarBlue 方法具有更高的评分者间一致性，并且在高资源和低资源环境下的实验室中，都可以提供一种更可靠的、标准化的方法。
