基质辅助激光解吸电离飞行时间质谱鉴定丝状真菌的影响因素分析

李颖; 张戈; 杨洋; 李冬雪; 王贺; 徐英春

doi:10.3969/j.issn.1674-9081.20170245

基质辅助激光解吸电离飞行时间质谱鉴定丝状真菌的影响因素分析

Evaluation of Influence Factors in the Identification of Clinical Filamentous Fungi by Matrix Assisted Laser Desorption Ionization-time of Flight Mass Spectrometry

摘要

摘要:
目的评估菌株培养条件、培养天数以及结果判定截断值(cut-off value, COV) 3种因素对基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption ionization-time of flight mass spectrometry, MALDI-TOF MS)鉴定丝状真菌能力的影响。
方法将78株受试丝状真菌分别接种到沙堡弱葡萄糖琼脂(sabouraud dextrose agar, SDA)、土豆葡萄糖琼脂(potato dextrose agar, PDA)两种培养基及28、35℃两种培养温度共4种组合的培养条件下进行培养, 并于培养第2、3、4、5天以及第7天对其进行MALDI-TOF MS分析, 根据鉴定率的差异筛选菌株MALDI-TOF MS鉴定用最佳培养条件和培养天数; 下调MALDI-TOF MS结果判定COV, 分析其对MALDI-TOF MS丝状真菌鉴定率的影响。
结果受试菌株在SDA和PDA两种培养基间的MALDI-TOF MS鉴定率无显著性差异(χ²=0.467, P=0.792), 但28℃培养温度下的鉴定率高于35℃培养温度(χ²=7.195, P=0.027);培养第2、3天种水平(40.3%和34.3%)和属水平(44.8%和46.3%)鉴定率均较高; 将MALDI-TOF MS种水平判定COV由 > 2.0下调至 > 1.7后, 受试菌株种水平判定率显著提升(85.9%比32.1%, χ²=40.119, P < 0.01)且不增加错误鉴定率。
结论丝状真菌以SDA或PDA培养基28℃培养2~3 d, 并以分值> 1.7作为种水平判定COV, 可有效提高MALDI-TOF MS对丝状真菌的鉴定能力。

Abstract:
Objective This study aimed to evaluate the potential influence of culture conditions, incubation time, and the interpreting cut-off value(COV) in the identification of filamentous fungi by matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS).
Methods Seventy-eight isolates of filamentous fungi were cultivated in sabouraud dextrose agar(SDA) or potato dextrose agar(PDA) media with an incubation temperature of 28℃ or 35℃ separately, namely four different culture conditions.MALDI-TOF MS identification was carried out at different time including the 2nd, 3rd, 4th, 5th and 7th days of incubation of the isolates. Differences in identification capacities of MALDI-TOF MS under the tested parameters were analyzed to determine the optimal culture condition and incubation time of filamentous fungi. The influence of lowered interpreting COV in MALDI-TOF MS identification of filamentous fungi was also evaluated.
Results No significant difference was detected between the SDA and PDA culture media in the identification capacities of MALDI-TOF MS(χ²=0.467, P=0.792). The identification capacity of MALDI-TOF MS with the incubation temperature of 28℃ was superior to that of 35℃(χ²=7.195, P=0.027). Higher identification rates of species level(40.3%, 34.3%) and genus level(44.8%, 46.3%) were achieved in isolates incubated for 2 and 3 days. The identification rate of species level was increased significantly from 32.1% to 85.9%(χ²=40.119, P < 0.01) without increasing the false identification rate after adjusting the interpreting COV from score > 2.0 to > 1.7.
Conclusion The performance of MALDI-TOF MS in the identification of filamentous fungi could be improved by applying the optimal culture condition of incubating in SDA or PDA media under the temperature of 28℃ for 2 to 3 days and by adjusting the interpreting COV of the species level with a score > 1.7.

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