MALDI-TOF MS In Rapid Identification Of Invasive Filamentous Fungi

Objective: To investigate the influence of different culture media,different culture time and different protein extraction methods on the accuracy of mass spectrometry identification of invasive filamentous fungi.This study aimed to improve the accuracy of MALDI-TOF MS in the identification of invasive filamentous fungi.Methods: Traditional morphological method and MALDI-TOF MS were used to identify clinical filamentous fungi,meanwhile,molecular identification was used as the standard method.According to the identification results of molecular sequencing,the strains that were not included in VITEK-MS V3.0 database were removed,and the rest strains were inoculated in three different culture media(SDA,PDA and CA).The specific spectral fingerprints of different culture time points(2,3,5,7and 9 days)were obtained by using two different protein extraction methods(extracting protein with formic acid and acetonitrile,extracting protein with magnetic bead,formic acid and acetonitrile).Results: Comparison of different protein extraction methods of filamentous fungi,the total identification accuracy of extracting protein with formic acid and acetonitrile was79.8%,the total identification accuracy of extracting protein with magnetic bead,formic acid and acetonitrile was 77.5%.There was no significant difference between two different protein extraction methods for mass spectrometry(? 2 = 1.040,P = 0.308).Comparison of different media showed that the total identification accuracy of SDA medium,PDA medium and CA medium were 90.7%,81.4%,and 67.4%,respectively.There was significant difference among the three different culture media(? 2 = 36.609,P= 0.000).Among these media,SDA medium was the highest identification accuracy and CA medium was the lowest(SDA vs PDA,? 2 = 7.748,P = 0.005;SDA VS CA,? 2 =35.131,P = 0.000;PDA VS CA,? 2 = 10.994,P = 0.001).Comparison of different culturetime,the total identification accuracy of different culture time points(2,3,5,7and 9days)were 64.3%,88.4%,89.1%,79.1% and 78.3% respectively.There was significant difference among the five different culture time(?2 = 32.274,P = 0.000).The identification accuracy of mass spectrometry on the third and fifth day of culture was better than that on the seventh and ninth day of culture.In addition,the identification accuracy of mass spectrometry on the second day of culture was significantly lower than that on the other days(3 days vs 5 days,? 2 = 0.039,P = 0.844;7 days vs 9 days,?2 = 0.023,P = 0.879;3 days vs 7 days,? 2 = 4.095,P = 0.043;2 days vs 9 days,? 2 =6.139,P = 0.013).Conclusion: The best procedure to identify invasive filamentous fungi by mass spectrometry is to culture them on SDA for 3 days and then extract them with formic acid and acetonitrile.