李颖;张戈;杨洋;李冬雪;王贺;徐英春;
LI Ying;ZHANG Ge;YANG Yang;LI Dong-xue;WANG He;XU Ying-chun;Department of Clinical Laboratory,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College;Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College;

• 1:中国医学科学院北京协和医学院北京协和医院检验科
• 2:中国医学科学院北京协和医学院北京协和医院侵袭性真菌病精准诊断与机制研究北京市重点实验室

摘要(Abstract)：

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(χ~2=0.467,P=0.792),但28℃培养温度下的鉴定率高于35℃培养温度(χ~2=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%,χ~2=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。
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 2 nd, 3 rd, 4 th, 5 th and 7 th 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. ResultsNo significant difference was detected between the SDA and PDA culture media in the identification capacities of MALDI-TOF MS(χ~2=0.467, P=0.792). The identification capacity of MALDI-TOF MS with the incubation temperature of 28 ℃ was superior to that of 35 ℃(χ~2=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%(χ~2=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.

关键词(KeyWords)： 基质辅助激光解吸电离飞行时间质谱;丝状真菌;鉴定;影响因素
matrix assisted laser desorption ionization-time of flight mass spectrometry;filamentous fungi;identification;influence factors

Abstract:

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基金项目(Foundation):

作者(Author): 李颖;张戈;杨洋;李冬雪;王贺;徐英春;
LI Ying;ZHANG Ge;YANG Yang;LI Dong-xue;WANG He;XU Ying-chun;Department of Clinical Laboratory,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College;Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College;

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参考文献(References)：

• [1] Taccone FS,Van den Abeele AM,Bulpa P,et al.Epidemiology of invasive aspergillosis in critically ill patients:clinical presentation,underlying conditions,and outcomes[J].Crit Care,2015,19:7.
• [2] Liao Y,Chen M,Hartmann T,et al.Epidemiology of opportunistic invasive fungal infections in China:review of literature[J].Chin Med J(Engl),2013,126:361- 368.
• [3] Slavin M,van Hal S,Sorrell TC,et al.Invasive infections due to filamentous fungi other than Aspergillus:epidemiology and determinants of mortality[J].Clin Microbiol Infect,2015,21:490.e1-e10.
• [4] Blum G,H?rtnagl C,Jukic E,Erbeznik T,et al.New insight into amphotericin B resistance in Aspergillus terreus[J].Antimicrob Agents Chemother,2013,57:1583- 1588.
• [5] Martos AI,Romero A,González MT,et al.Evaluation of the Etest method for susceptibility testing of Aspergillus spp.and Fusarium spp.to three echinocandins[J].Med Mycol,2010,48:858- 861.
• [6] Alastruey-Izquierdo A,Castelli MV,Cuesta I,et al.In vitro activity of antifungals against Zygomycetes[J].Clin Microbiol Infect,2009,15:71- 76.
• [7] Halliday CL,Chen SC,Kidd SE,et al.Antifungal susceptibilities of non-Aspergillus filamentous fungi causing invasive infection in Australia:support for current antifungal guide-line recommendations[J].Int J Antimicrob Agents,2016,48:453- 458.
• [8] Singhal N,Kumar M,Kanaujia PK,et al.MALDI-TOF mass spectrometry:an emerging technology for microbial identification and diagnosis[J].Front Microbiol,2015,6:791.
• [9] Cassagne C,Normand AC,L'Ollivier C,et al.Performance of MALDI-TOF MS platforms for fungal identification[J].Mycoses,2016,59:678- 690.
• [10] Schulthess B,Ledermann R,Mouttet F,et al.Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory[J].J Clin Microbiol,2014,52:2797- 2803.
• [11] Lau AF,Drake SK,Calhoun LB,et al.Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by MALDI-TOF MS[J].J Clin Microbiol,2013,51:828- 834.
• [12] Li Y,Wang H,Zhao YP,et al.Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry system for identification of Aspergillus species directly from growth on solid agar media[J].Front Microbiol,2017,8:1209.
• [13] 李颖,徐英春.评价ITS、BenA 和CaM 序列分析在曲霉菌种鉴定方面的应用[J].中国真菌学杂志,2017,12:74- 77.
• [14] 李颖,郭莉娜,徐英春.内转录间隔区(ITS)序列分析技术对丝状真菌临床分离株鉴定能力的评估[J].中华微生物学和免疫学杂志,2017,37:607- 610.
• [15] Alanio A,Beretti JL,Dauphin B,et al.MALDI-TOF MS for fast and accurate identification of clinically relevant Aspergillus species[J].Clin Microbiol Infect,2011,17:750- 755.
• [16] Samson RA,Visagie CM,Houbraken J,et al.Phylogeny,identification and nomenclature of the genus Aspergillus[J].Stud Mycol,2014,78:141- 173.