探讨质谱（MALDI-TOF MS）在临床分离的丝状真菌快速鉴定中的应用。

收集首都医科大学宣武医院2017年1月至2019年1月经形态学鉴定为丝状真菌的菌株，分别采用双甲酸夹心法和甲酸乙腈萃取法提取真菌蛋白并行MALDI-TOF MS鉴定，并与聚合酶链反应（PCR）内转录间隔区（ITS）基因序列测定结果比较；分析丝状真菌菌种分布和标本来源。

共收集临床分离的丝状真菌90株，其中曲霉菌属70株（77.78%），青霉菌属6株（6.67%），链格孢霉5株（5.56%），镰刀菌属4株（4.44%），赛多孢菌2株（2.22%），米根霉、根毛霉和哈茨木霉各1株（1.11%）；其中曲霉菌以烟曲霉47株（52.22%）、黄曲霉13株（14.44%）和黑曲霉4株（4.44%）为主。标本主要来源于呼吸道标本（90.0%）（包括痰标本和肺泡灌洗液），其次为角膜和耳道分泌物（3.33%），痰标本中分离率最多的为曲霉菌（59株、65.56%）。MALDI-TOF MS鉴定到种水平82株，与基因测序结果一致率为91.11%（82/90），鉴定准确率显著高于形态学鉴定结果（68.89%，62/90），差异有统计学意义（χ² = 12.02、P = 0.01）；质谱鉴定丝状真菌中的曲霉菌、镰刀菌、赛多孢菌和链格孢霉时准确率高（98.57%～100%）；双甲酸夹心法和甲酸乙腈萃取法前处理的质谱鉴定丝状真菌到种水平的比率分别为91.11%（82/90）和90.00%（81/90），鉴定曲霉菌种水平分别为98.57%（69/70）和97.14%（68/70），但鉴定准确率差异无统计学意义（χ² = 0.34、P = 0.95）；两种前处理方法均可100%鉴定烟曲霉、黄曲霉、赛多孢菌和链格孢霉到种水平，其中89.36%（42/47）烟曲霉和76.92%（10/13）黄曲霉鉴定得分≥ 2.0分。

MALDI-TOF MS技术鉴定丝状真菌快速准确，双甲酸夹心法前处理操作更简便，适合实验室常规检测，可用于实验室常规鉴定曲霉菌等常见丝状真菌。

To investigate the application value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in rapid identification of clinical filamentous fungi.

The strains identified as filamentous fungi by morphology from January 2017 to January 2019 in Xuanwu Hospital of Capital Medical University were collected and identified by MALDI-TOF MS with the method of double formic acid sandwich and formic acid acetonitrile extraction. The results were compared with the results of its gene sequencing of polymerase chain reaction (PCR) in internal transcribed spacer (ITS). The distribution and source of filamentous fungi were analyzed.

A total of 90 clinical isolates of filamentous fungi were collected, including 70 strains of Aspergillus spp. (77.78%), 6 strains of Penicillium spp. (6.67%), 5 strains of Alternaria (5.56%), 4 strains of Fusarium spp. (4.44%), 2 strains of Sedosporium spp. (2.22%), 1 strain each of Rhizopus, Mucor and Trichoderma harzianum (1.11%). Aspergillus fumigatus (47 strains, 52.22%), Aspergillus flavus (13 strains, 14.44%) and Aspergillus niger (4 strains, 4.44%) were the main isolates. The specimens were mainly from respiratory tract (90.0%) (including sputum and alveolar lavage fluid), followed by cornea and ear canal secretion (3.33%), of which Aspergillus spp. were the mainly strains isolated from sputum samples (59 strains, 65.56%). Compared with the results of gene sequencing, the accuracy of MALDI-TOF MS was significantly higher than that of morphology (91.11% vs. 68.89%; χ² = 12.02, P = 0.01). MALDI-TOF MS was used to identify Aspergillus spp., Fusarium spp., Sedosporium spp. and Alternaria with high accuracy (98.57%-100%). The identification rates of filamentous fungi to species level were 91.11% (82/90) and 90% (81/90) by MALDI-TOF MS pretreated with dicarboxylic acid sandwich method and formic acid acetonitrile extraction method, and the identification rates of aspergillus species were 98.57% (69/70) and 97.14% (68/70), respectively. There was no significant difference in the identification accuracy of the two pretreatment methods (χ² = 0.34, P = 0.95). Both pretreatment methods could identify 100% Aspergillus fumigata, Aspergillus aflatus, Sedosporium and Alternaria spp. Among them, 89.36% (42/47) Aspergillus fumigatus and 76.92% (10/13) Aspergillus flavus scored ≥ 2.0.

The MALDI-TOF MS technique was rapid and accurate in the identification of filamentous fungi, and the double formic acid sandwich pretreatment method was more convenient and suitable for routine laboratory detection. It could be used for routine laboratory identification of common filamentous fungi such as Aspergillus.