Postgraduate Program in Medicine: Medical Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
Department of Sciences, Humanities and Education, Postgraduate Program in Health Promotion, Postgraduate Program in Environmental Technology, Universidade de Santa Cruz do Sul, Santa Cruz do Sul, Rio Grande do Sul, Brazil.
PLoS One. 2020 Dec 2;15(12):e0243231. doi: 10.1371/journal.pone.0243231. eCollection 2020.
Fonsecaea pedrosoi is one of the main agents of chromoblastomycosis, a chronic subcutaneous mycosis. Itraconazole (ITC) is the most used antifungal in its treatment, however, in vitro antifungal susceptibility tests are important to define the best therapy. These tests are standardized by the Clinical and Laboratory Standards Institute (CLSI), but these protocols have limitations such as the high complexity, cost and time to conduct. An alternative to in vitro susceptibility test, which overcomes these limitations, is FTIR. This study determined the minimum inhibitory concentration (MIC) of itraconazole for F. pedrosoi, using FTIR and chemometrics. The susceptibility to ITC of 36 strains of F. pedrosoi was determined according to CLSI and with the addition of tricyclazole (TCZ), to inhibit 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Strains were grown in Sabouraud agar and prepared for Attenuated Total Reflection (ATR)/FTIR. Partial least squares (PLS) regression was performed using leave-one-out cross-validation (by steps of quintuplicates), then tested on an external validation set. A coefficient of determination (R²) higher than 0.99 was obtained for both the MIC-ITC and MIC-ITC+TCZ ATR/PLS models, confirming a high correlation of the reference values with the ones predicted using the FTIR spectra. This is the first study to propose the use of FTIR and chemometric analyses according to the M38-A2 CLSI protocol to predict ITC MICs of F. pedrosoi. Considering the limitations of the conventional methods to test in vitro susceptibility, this is a promising methodology to be used for other microorganisms and drugs.
佩德罗索外瓶霉是黑曲霉病（一种慢性皮下真菌感染）的主要病原体之一。伊曲康唑（ITC）是治疗该病最常用的抗真菌药物，但体外抗真菌药敏试验对于确定最佳治疗方案至关重要。这些试验由临床和实验室标准协会（CLSI）标准化，但这些方案存在一些局限性，如操作复杂、成本高和耗时。FTIR 是一种替代体外药敏试验的方法，它克服了这些局限性。本研究采用 FTIR 和化学计量学方法测定了伊曲康唑对佩德罗索外瓶霉的最小抑菌浓度（MIC）。根据 CLSI 标准和添加三唑酮（TCZ）抑制 1,8-二羟基萘（DHN）-黑色素生物合成，测定了 36 株佩德罗索外瓶霉对 ITC 的敏感性。将菌株在沙氏琼脂上生长并制备用于衰减全反射（ATR）/FTIR。使用留一法五重交叉验证（逐步五倍）进行偏最小二乘（PLS）回归，然后在外部验证集上进行测试。MIC-ITC 和 MIC-ITC+TCZ ATR/PLS 模型的决定系数（R²）均高于 0.99，证实了参考值与使用 FTIR 光谱预测的值之间具有高度相关性。这是第一项根据 CLSI M38-A2 方案提出使用 FTIR 和化学计量分析来预测 F. pedrosoi ITC MIC 的研究。考虑到传统方法在体外药敏试验方面的局限性，这是一种很有前途的方法，可用于其他微生物和药物。
