MALDI-TOF MS对临床侵袭性丝状真菌的快速鉴定技术的研究

Rapid identification technology of invasive filamentous fungi by MALDI-TOF MS

目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术在临床常见丝状真菌鉴定中的应用价值。方法收集2017年4月—2019年7月本院各种类型临床标本中分离培养的65株丝状真菌,以及某三甲医院惠赠的26株丝状真菌。这91株丝状真菌通过传统形态学鉴定、DNA测序及MALDI-TOF MS鉴定方法后,以DNA测序结果为标准,与另外2种方法的鉴定结果进行统计学比较,评价MALDI-TOF MS在临床丝状真菌鉴定上的应用。结果在28℃和35℃条件下培养的菌株经质谱仪鉴定后,χ^(2)检验显示P>0.05,提示培养温度差异对结果的影响无统计学意义;以DNA测序结果为金标准,传统形态学方法的正确鉴定率为81.3%(74/91),而基于MALDI-TOF MS技术的正确鉴定率可达97.8%(89/91);研究中,使用“甲酸乙腈提取法”提取蛋白后,丝状真菌质谱鉴定的正确率为90.1%(82/91),而“磁珠研磨法”提取蛋白后的质谱鉴定率则为97.8%(89/91)。结论临床常规工作中使用质谱仪鉴定常见丝状真菌时,培养温度不会影响质谱鉴定结果;改良“磁珠研磨法”提取蛋白后质谱仪鉴定的正确率优于质谱仪推荐“甲酸乙腈提取法”;MALDI-TOF MS技术在常见丝状真菌的鉴定上,相比于传统形态学鉴定,该技术更快速、客观、高效及准确。

Objective To evaluate the application value of matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS) technology in the identification of clinical common filamentous fungi in clinical practice. Methods From April 2017 to July 2019, 65 filamentous fungi were isolated and cultured from various types of clinical specimens of ourhospital and 26 common filamentous fungi donated by a large third-class hospital. After the 91 filamentous fungi were identified by traditional morphological identification, DNA sequencing and MALDI-TOF MS, the results of DNA sequencing were compared with those of the other two methods to evaluate the application of MALDI-TOF MS in the identification of clinical filamentous fungi. Results The filamentous fungi cultured at 28℃ and 35℃ were identified by mass spectrometer, the Chi-square test(χ^(2) test, P=0.05) showed that P>0.05, suggesting that there was no significant difference in culture temperature between the two groups.Using DNA sequencing results as gold standard, the correct identification rate of traditional morphological methods was 81.3%(74/91), while that of MALDI-TOF MS technology was 97.8%(89/91). In this study, the correct identification rate of filamentous fungi by mass spectrometry was 90.1%(82/91) after extracting protein by acetonitrile formate extraction method, while that of protein extracted by magnetic bead grinding method was 97.8%(89/91). Conclusion In clinical routine work, when using mass spectrometer to identify common filamentous fungi,the culture temperature will not affect the mass spectrometry identification results;the correctness of mass spectrometry after improved magnetic bead grinding method to extract protein is better than that recommended by mass spectrometer to extract acetonitrile formate;compared with traditional morphological identification,MALDI-TOF MS technology is faster,more objective,more efficient and more accurate in identification of common filamentous fungi.