MALDI-TOF MS in rapid identification of invasive Filamentous fungi.
Objective: To investigate the influence of different culture media, culture time and protein extraction methods on the accuracy of mass spectrometry(MS) identification of invasive Filamentous fungi(IFF), and improve the accuracy of MALDI-TOF MS in the identification of IFF. Methods: MALDI-TOF MS was used to identify clinical Filamentous fungi; 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 remaining strains were inoculated in three different culture media(SDA, PDA and CA). The specific spectral fingerprints at different culture time points(2, 3, 5, 7 and 9 days) were obtained by using two different protein extraction methods(extraction with formic acid and acetonitrile; extraction with magnetic bead, formic acid and acetonitrile). Results: Comparison of different protein extraction methods showed that the total identification accuracy for extraction with formic acid and acetonitrile was 79.8%, while that with magnetic bead, formic acid and acetonitrile was 77.5%; there was no significant difference between the two extraction methods(χ2=1.040, P=0.308). Comparison of different media showed that the total identification accuracies for SDA medium, PDA medium and Ca medium were 90.7%, 81.4% and 67.4%, respectively. There were significant differences among the three culture media(χ2=36.609, P<0.001). SDA medium resulted in the highest identification accuracy and Ca medium did the lowest(SDA vs PDA, χ2=7.748, P=0.005; SDA vs CA, χ2=35.131, P<0.001; PDA vs CA, χ2=10.994, P=0.001). Comparison of different culture time point showed that the total identification accuracies at Day 2, 3, 5, 7 and 9 were 64.3%, 88.4%, 89.1%, 79.1% and 78.3%, respectively; there were significant differences among the five culture time points(χ2=32.274, P<0.001). The identification accuracies of MS on the third and fifth day of culturing were better than those on the seventh and ninth day. In addition, the identification accuracy of MS at Day 2 of culturing was significantly lower than those at other days(3 days vs 5 days, χ2=0.039, P=0.844; 7 days vs 9 days, χ2=0.023, P=0.879; 3 days vs 7 days, χ2=4.095, P=0.043; 2 days vs 9 days, χ2=6.139, P=0.013). Conclusion: The best procedure to identify invasive Filamentous fungi with mass spectrometry is to culture them on SDA for 3 days and then extract them with formic acid and acetonitrile.