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Matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry: Protocol standardisation, comparison and database expansion for faster and reliable identification of dermatophytes
Author(s) -
Shaw Dipika,
Ghosh Anup K.,
Paul Saikat,
Singh Shreya,
Chakrabarti Arunaloke,
Kaur Harsimran,
Narang Tarun,
Dogra Sunil,
Rudramurthy Shivaprakash M.
Publication year - 2021
Publication title -
mycoses
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.13
H-Index - 69
eISSN - 1439-0507
pISSN - 0933-7407
DOI - 10.1111/myc.13285
Subject(s) - matrix assisted laser desorption/ionization , dendrogram , extraction (chemistry) , mass spectrometry , dna extraction , chromatography , biology , chemistry , medicine , polymerase chain reaction , genetics , desorption , organic chemistry , adsorption , gene , population , environmental health , genetic diversity
Background Accurate and early identification of dermatophytes enables prompt antifungal therapy. However, phenotypic and molecular identification methods are time‐consuming. MALDI‐TOF MS‐based identification is rapid, but an optimum protocol is not available. Objectives To develop and validate an optimum protein extraction protocol for the efficient and accurate identification of dermatophytes by MALDI‐TOF MS. Materials/methods Trichophyton mentagrophytes complex ( n = 4), T. rubrum ( n = 4) and Microsporum gypseum ( n = 4) were used for the optimisation of protein extraction protocols. Thirteen different methods were evaluated. A total of 125 DNA sequence confirmed clinical isolates of dermatophytes were used to create and expand the existing database. The accuracy of the created database was checked by visual inspection of MALDI spectra, MSP dendrogram and composite correlation index matrix analysis. The protocol was validated further using 234 isolates. Result Among 13 protein extraction methods, six correctly identified dermatophytes but with a low log score (≤1.0). The modified extraction protocol developed provided an elevated log score of 1.6. Significant log score difference was observed between the modified protocol and other existing protocols ( T. mentagrophytes complex: 1.6 vs. 0.2–1.0, p < .001; T. rubrum : 1.6 vs. 0.4–1.0, p < .001; M. gypseum :1.6 vs. 0.2–1.0, p < .001). Expansion of the database enabled the identification of all 234 isolates (73.5% with log score ≥2.0 and 26.4% with log scores range: 1.75–1.99). The results were comparable to DNA sequence‐based identification. Conclusion MALDI‐TOF MS with an updated database and efficient protein extraction protocol developed in this study can identify dermatophytes accurately and also reduce the time for identifying them.