TWO PROTEIN EXTRACTION METHODS FOR THE Candida SPECIES IDENTIFICATION BY MALDI-TOF

The Candida species are responsible for the large part of nosocomial fungal infections and have great medical relevance. Thus, the rapid and correct etiological agent identification is essential to control the mortality rates. However, currently available laboratory techniques are mostly time-consuming and labor-intensive. The matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) has emerged as new technique for the microorganisms identification simple, correct fast and low operation cost. Therefore, the aim of this study was to evaluate two technique of protein extraction for the identification of Candida species by MALDI-TOF MS system VITEK MS (bioMérieux). Several clinical isolates of Candida albicans (11 isolates), Candida glabrata (6), Candida tropicalis (5), Candida parapsilosis (4) and Candida krusei (5) contained in the Micoteca of Medical Mycology Laboratory were evaluated, of State University of Maringá. The isolates were cultivated in CHROMagar at 35ºC for 48 hours. For the simple extraction with formic acid (FA), a single colony were added with 0,5µL of FA in the target plate.After dry, 1µL of α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (bioMérieux) was added and dried at room temperature. For the extraction with FA/ACN, in one microtube were added 300µL of distilled water, a single colony and 900µL of ethanol. The mixed was centrifuged at 13,400 rpm for 2 min. The pellet was reconstituted in 50µL of FA (70%). After vortex, 50µL of acetonitrile was added. Again, was centrifuged at 13,400 rpm for 2 min. Finally, 1µL of the supernatant was inserted in the plate and after drying at ambient temperature, 1µL HCCA be added.. The sample information was transferred to the VITEK MS acquisition station using Myla v3.2 Middleware. The results were automatically calculated and presented by using VITEK MS IVD Knowledgebase v.2.0, and it was reported as a confidence value up to 90.0%. The most of Candida species were correctly identified by extraction with FA, except C tropicalis (0%). While the extraction with FA/ACN was effective in identifying of 100% of C. tropicalis and C. krusei isolates, 81.1% C. albicans isolates and 75% C. parapsilosis. The last methodology was not able to identify any of the C. glabrata isolates. Thus, this work shows that the protein extraction process is essential for the correct identification of Candida species. The simple extraction with FA favored the correct diagnosis of C. glabrata species, but it was not enough to allow protein extraction and identification of C. tropicalis, requiring extraction more elaborate