MALDI-TOF ICMS: capability, potentiality and limits in the fast identification of Trichophyton rubrum from clinical cases occurrence in Portuguese health centres

Abstract

Objective: Trichophyton rubrum is presently the most common worldwide pathogen causing dermatophytoses such as tinea corporis, tinea capitis, tinea pedis, and onychomycosis [1]. The main aim of the present work was assess MALDI-TOF ICMS as a fast and reliable technique in the identification of T. rubrum from clinical cases occurrence in the Portuguese health centres, and evaluates the potentialities and limits of this new microbial identification technique on the taxonomy of these infectious dermatophytes. Methods: Fungi were grown for 10 days in solid medium (SDA, Sabouraud Dextrose Agar) and then the mycelia were direct transferred from the SDA plate to the MALDI stainless steel template and mixed with 1 ml MALDI matrix solution (75 mg/ml 2,5-dihydroxybenzoic acid in ethanol/water/acetonitrile [1:1:1] with 0.03% trifluoroacetic acid). The sample mixtures were air dried at room temperature. The analyses were performed in our laboratory on an Axima LNR system (Kratos Analytical, Shimadzu, Manchester, UK) equipped with a nitrogen laser (337 nm). The mass range from m/z = 2,000 to 20,000 Da was recorded. Escherichia coli strain DH5a with known mass values of ribosomal proteins was used for external calibration. The fungi classification was performed on the SARAMIS software (AnagnosTec mbH, Potsdam- Golm, Germany). Molecular biology was used when appropriated with PCR based-technology. The presence of a 203-bp PCR product confirmed T. rubrum identification. Results: All strains were accurately and consistently identified as T. rubrum by MALDI-TOF ICMS combined to SARAMIS database analysis. Spectral mass analysis proven to be a rapid method since the analysis took only a few minutes to perform with the benefit of any laborious sample preparation procedures or any expensive chemical reagent was needed. Conclusions: The fungal spectral analysis by MALDI-TOF ICMS was as good as molecular biology in order to identify T. rubrum but much faster and cheaper.