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Abstract：
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.
We developed a colorimetric alamarBlue method to determine the susceptibility of antifungal drugs against Talaromyces marneffei. We observed excellent agreement between the alamarBlue method and the Clinical Laboratory Standard Institute broth microdilution method, and the alamarBlue method had substantially higher inter-rater agreement.
We developed a colorimetric alamarBlue method to determine the susceptibility of antifungal drugs against Talaromyces marneffei. We observed excellent agreement between the alamarBlue method and the Clinical Laboratory Standard Institute broth microdilution method, and the alamarBlue method had substantially higher inter-rater agreement.
摘要：
抗真菌药物耐药性是侵袭性真菌感染治疗失败的新原因。和抗真菌药敏试验（AFST）可以告知治疗决定。目前,对于马尔尼菲塔拉菌（Tm）或其他双态真菌，尚无既定的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，与细胞代谢活动成比例地从蓝色变为粉红色。我们确定添加alamarBlue的最佳时间为接种后24小时，MIC读数为接种后72小时。我们的方法允许三种方法来确定最小抑制浓度（MIC）：视觉检查颜色变化，光密度,和荧光强度。我们通过确定7种抗真菌药物对32种Tm临床分离株的MIC来验证该测定，并评估了我们的alamarBlue与临床实验室标准研究所（CLSI）肉汤微量稀释方法之间的基本一致性和评估者间的可靠性。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，泊沙康唑为0.03-0.13μg/mL，对于氟胞嘧啶，0.06-0.5µg/mL，两性霉素B为0.5-1µg/mL，卡泊芬净0.5-4微克/毫升，和0.5-16µg/mL的氟康唑。AlamarBlue方法的所有三个MIC读数之间的基本协议是100%，AlamarBlue和CLSI方法之间的94%到100%。与已建立的CLSI方法相比，我们的alamarBlue方法具有更高的评分者间协议，并提供了一种更可靠的方法，可以在高资源和低资源环境中跨实验室进行标准化。
我们开发了一种比色alamarBlue方法来确定抗真菌药物对马尔尼菲塔拉霉素的敏感性。我们观察到alamarBlue方法和临床实验室标准研究所肉汤微量稀释方法之间的极好一致性，和alamarBlue方法具有更高的评分者之间的一致性。
我们开发了一种比色alamarBlue方法来确定抗真菌药物对马尔尼菲塔拉霉素的敏感性。我们观察到alamarBlue方法和临床实验室标准研究所肉汤微量稀释方法之间的极好一致性，和alamarBlue方法具有更高的评分者之间的一致性。
