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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

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文章导航 > 协和医学杂志  > 2020 >  11(2): 156-161
李颖, 张戈, 杨洋, 李冬雪, 王贺, 徐英春. 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[J]. 协和医学杂志, 2020, 11(2): 156-161. doi: 10.3969/j.issn.1674-9081.20170245
引用本文: 李颖, 张戈, 杨洋, 李冬雪, 王贺, 徐英春. 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[J]. 协和医学杂志, 2020, 11(2): 156-161. doi: 10.3969/j.issn.1674-9081.20170245
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Ying LI, Ge ZHANG, Yang YANG, Dong-xue LI, He WANG, Ying-chun XU. Evaluation of Influence Factors in the Identification of Clinical Filamentous Fungi by Matrix Assisted Laser Desorption Ionization-time of Flight Mass Spectrometry[J]. Medical Journal of Peking Union Medical College Hospital, 2020, 11(2): 156-161. doi: 10.3969/j.issn.1674-9081.20170245
Citation: Ying LI, Ge ZHANG, Yang YANG, Dong-xue LI, He WANG, Ying-chun XU. Evaluation of Influence Factors in the Identification of Clinical Filamentous Fungi by Matrix Assisted Laser Desorption Ionization-time of Flight Mass Spectrometry[J]. Medical Journal of Peking Union Medical College Hospital, 2020, 11(2): 156-161. doi: 10.3969/j.issn.1674-9081.20170245
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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

doi: 10.3969/j.issn.1674-9081.20170245
  • 1.

    中国医学科学院 北京协和医学院 北京协和医院 检验科 北京市重点实验室, 北京 100730

  • 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, 北京 100730

详细信息
    通讯作者:

    徐英春 电话:010-69159766, E-mail:xycpumch@139.com

  • 中图分类号: R446.5

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

  • 1.

    Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China

  • 2.

    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, Beijing 100730, China

More Information
    Corresponding author: XU Ying-chun Tel: 86-10-69159766, E-mail: xycpumch@139.com

    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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  • 图  1  MALDI-TOF MS鉴定丝状真菌的菌落取材部位(A，方框)和取材量(B)

    MALDI-TOF MS:基质辅助激光解吸电离飞行时间质谱

    下载: 全尺寸图片 幻灯片

    表  1  菌株培养条件对MALDI-TOF MS鉴定丝状真菌能力的影响[n(%)]

    菌种 株数 SDA PDA
    28 ℃ 35 ℃ 28 ℃ 35 ℃
    种水平 属水平 无法鉴定 种水平 属水平 无法鉴定 种水平 属水平 无法鉴定 种水平 属水平 无法鉴定
    曲霉 67 21(31.3) 31(42.3) 15(22.4) 15(22.4) 32(47.8) 20(29.8) 19(28.4) 34(50.7) 14(20.9) 14(20.9) 34(50.7) 19(28.4)
      烟曲霉 39 7 23 9 6 21 12 9 21 9 4 23 12
      黄曲霉 13 7 4 2 5 5 3 5 7 1 6 5 2
      黑曲霉 7 3 1 3 2 2 3 2 3 2 1 3 3
      土曲霉 4 1 2 1 0 2 2 1 1 2 1 1 2
      构巢曲霉 3 3 0 0 2 1 0 2 1 0 2 1 0
      杂色曲霉 1 0 1 0 0 1 0 0 1 0 0 1 0
    镰刀菌 7 1(14.3) 4(57.1) 2(28.6) 0(0) 2(28.6) 5(71.4) 1(14.3) 5(71.4) 1(14.3) 0(0) 2(28.6) 5(71.4)
      茄病镰刀菌 3 1 1 1 0 1 2 1 2 0 0 1 2
      串珠镰刀菌 3 0 2 1 0 1 2 0 2 1 0 0 3
      层生镰刀菌 1 0 1 0 0 0 1 0 1 0 0 1 0
    接合菌 4 0(0) 2(50.0) 2(50.0) 0(0) 0(0) 4(100) 0(0) 1(25.0) 3(75.0) 0(0) 1(25.0) 3(75.0)
      小孢根霉 2 0 1 1 0 0 2 0 0 2 0 0 2
      卷枝毛霉 1 0 1 0 0 0 1 0 1 0 0 1 0
      总状共头霉 1 0 0 1 0 0 1 0 0 1 0 0 1
    总计 78 22(28.2) 37(47.4) 19(24.4) 15(19.3) 34(43.6) 29(37.2) 20(25.6) 40(51.3) 18(23.1) 14(17.9) 37(47.4) 27(34.6)
    MALDI-TOF MS：同图 1；SDA:沙堡弱葡萄糖琼脂；PDA:土豆葡萄糖琼脂
    下载: 导出CSV

    表  2  培养天数对MALDI-TOF MS鉴定丝状真菌能力的影响[n(%)]

    菌种 株数 第2天 第3天 第4天 第5天 第7天
    种水平 属水平 种水平 属水平 种水平 属水平 种水平 属水平 种水平 属水平
    曲霉 67 27(40.3) 30(44.8) 23(34.3) 31(46.3) 9(13.4) 30(44.8) 3(4.5) 21(31.3) 0(0) 8(11.9)
      烟曲霉 39 12 21 10 21 5 15 0 11 0 2
      黄曲霉 13 6 5 6 4 2 5 1 5 0 3
      黑曲霉 7 4 2 3 3 0 5 0 2 0 0
      土曲霉 4 1 2 0 3 0 3 0 1 0 0
      构巢曲霉 3 3 0 3 0 2 1 2 1 0 3
      杂色曲霉 1 1 0 1 0 0 1 0 1 0 0
    镰刀菌 7 3(42.9) 4(57.1) 2(28.6) 5(71.4) 1(14.3) 4(57.1) 0(0) 4(57.1) 0(0) 3(42.9)
      茄病镰刀菌 3 2 1 1 2 0 2 0 2 0 1
      串珠镰刀菌 3 1 2 1 2 1 1 0 2 0 2
      层生镰刀菌 1 0 1 0 1 0 1 0 0 0 0
    接合菌 4 0(0) 4(100) 0(0) 4(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0)
      小孢根霉 2 0 2 0 2 0 0 0 0 0 0
      卷枝毛霉 1 0 1 0 1 0 0 0 0 0 0
      总状共头霉 1 0 1 0 1 0 0 0 0 0 0
    总计 78 30(38.5) 38(48.7) 25(32.1) 40(51.8) 10(12.8) 34(43.6) 3(3.8) 25(32.1) 0(0) 11(14.1)
    MALDI-TOF MS：同图 1
    下载: 导出CSV

    表  3  结果判定截断值对MALDI-TOF MS丝状真菌鉴定能力的影响[n(%)]

    菌种 株数 Bruker推荐截断值 调整后截断值
    种水平(＞2.0) 属水平(1.7~2.0) 种水平(＞1.7) 属水平(1.4~1.7)
    曲霉属 67 23(34.3) 33(49.3) 56(83.6) 9(13.4)
      烟曲霉 39 10 23 33 5
      黄曲霉 13 6# 4 10* 3
      黑曲霉 7 3 3 6 0
      土曲霉 4 0 3 3 1
      构巢曲霉 3 3 0 3 0
      杂色曲霉 1 1 0 1 0
    镰刀菌 7 2(28.6) 5(71.4) 7(100) 0(0)
      茄病镰刀菌 3 1 2 3 0
      串珠镰刀菌 3 1# 2 3# 0
      层生镰刀菌 1 0 1 1# 0
    接合菌 4 0(0) 4(100) 4(100) 0(0)
      小孢根霉 2 0 2 2 0
      卷枝毛霉 1 0 1 1# 0
      总状共头霉 1 0 1 1 0
    总计 78 25(32.1) 42(53.8) 67(85.9) 9(11.5)
    *其中2株黄曲霉被错误鉴定为米曲霉；#其中1株黄曲霉被错误鉴定为米曲霉，1株串珠镰刀菌被错误鉴定为茄病镰刀菌，1株层生镰刀菌被错误鉴定为串珠镰刀菌，1株卷枝毛霉被错误鉴定为多分枝毛霉
    下载: 导出CSV
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