Federal State Health Institution «Rostov-on-Don Anti-Plague Institute» of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare.
Klin Lab Diagn. 2022 Apr 17;67(4):244-249. doi: 10.51620/0869-2084-2022-67-4-244-249.
To carry the comparative analysis of sample preparation methods for the most effective identification of Candida yeast by mass spectrometric analysis. 265 strains of yeast and yeast-like fungi isolated from the sputum of patients with pneumonia were investigated. The selected strains were identified by conventional methods (cultural, morphological, tinctorial, enzymatic properties) and MALDI-ToF MS using the Autoflex speed III Bruker Daltonics mass spectrometer (Germany) and Flex Control software. To evaluate the effectiveness of fungi species determinination, the comparative analysis of sample preparation was performed using 4 methods: direct application to the target, an extended direct application method, protein extraction using ethanol/formic acid or trifluoroacetic acid. The accelerated scheme of identification of fungi by the culture method does not provide clear and unambiguous results. When using mass spectrometric analysis, the reliability of the results depended on the sample preparation. A comparative study of the effectiveness of fungi species determination by various methods of the sample preparation of 50 clinical isolates was carried out. It was revealed that the extraction of cells using TFC acid does not lead to the appearance of the recordable protein spectra. The use of direct and extended direct application methods made it possible to establish the species only in 32-44% of the strains. The most effective method of sample preparation was the method using formic acid and ethanol, which allowed us to determine the species affiliation in 100% of the studied fungi (Score 2.0). Depending on the yeast species, a high statistical indicator (Score≥2.3) was registered for 42-100% of samples. The results of present study show that the use of MALDI-ToF MS is the most reliable and informative method of Candida spp.identification.
为了通过质谱分析最有效地鉴定假丝酵母属酵母，进行了样品制备方法的比较分析。研究了从肺炎患者痰液中分离出的 265 株酵母和酵母样真菌。使用常规方法（培养、形态学、染色、酶特性）和 MALDI-ToF MS 鉴定所选菌株，使用 Autoflex speed III Bruker Daltonics 质谱仪（德国）和 Flex Control 软件。为了评估真菌种属鉴定的有效性，使用 4 种方法进行了样品制备的比较分析：直接应用于目标、扩展直接应用方法、使用乙醇/甲酸或三氟乙酸进行蛋白质提取。培养方法的真菌快速鉴定方案不能提供清晰和明确的结果。当使用质谱分析时，结果的可靠性取决于样品制备。对 50 株临床分离株的各种样品制备方法进行了真菌种属鉴定效果的比较研究。结果表明，使用 TFC 酸提取细胞不会导致可记录的蛋白质谱出现。直接和扩展直接应用方法仅在 32-44%的菌株中能够确定种属。最有效的样品制备方法是使用甲酸和乙醇的方法，该方法可使我们确定 100%研究真菌的种属归属（评分 2.0）。根据酵母种类，42-100%的样本记录了较高的统计指标（评分≥2.3）。本研究结果表明，MALDI-ToF MS 的使用是鉴定假丝酵母属最可靠和信息丰富的方法。
