Fast identification of dermatophytes by MALDI-TOF/MS

Fast identification of dermatophytes by MALDI-TOF/MS
Roozbeh Yalfani,^1,*
1. Department of Nursing, Faculty of Medical Science, Islamic Azad University,Varamin-Pishva branch, Tehran, Iran
Introduction: Classical identification of dermatophytes relies on culture characteristics, microscopic morphology, physiological tests and clinical data. Their overlapping phenotypic characteristics, however, may be confusing and the identification requires growth of the organisms in culture for at least one week, which delays the diagnosis. Molecular techniques allow a fast and reliable identification of dermatophytes. ITS (Internal Transcribed Spacer) DNA sequencing is presently considered the gold standard for molecular identification and phylogenetic analyses of dermatophytes. However, DNA sequencing is expensive and at least 2–3 days are required before a sequence is obtained from a culture. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now used routinely in clinical diagnostic laboratories as it is faster than PCR and requires little sample handling. It is a cost-effective and reliable technique for the identification and typing of microbial pathogens including bacteria, yeasts, and filamentous fungi. MALDI-TOF MS has already been used for the identification of dermatophytes, either after their isolation and cultivation or directly from clinical material.
Methods: The approach is based on the acquisition of a protein profile (between 2 and 20 kDa) and its comparison to a database with reference spectra. This MALDI-TOF MS-based identification technique analyzes the protein content from treated or intact cells of microorganisms under the form of a spectrum that is considered as a protein fingerprint specific of a micro-organism. An unknown microorganism is identified by comparing its spectrum with the spectra in the reference library. Four different MALDI-TOF MS benchtop platforms are now commercialized for the routine identification of fungi in clinical microbiology laboratories. Together with the Andromas (Andromas SAS, Paris, France) and the Axima@Saramis (Shimadzu/AnagnosTec, Duisburg, Germany) systems, the Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and the Vitek MS (bioMerieux, Marcy l’Etoile, France) systems are currently employed in Europe, whereas only the latter two systems are approved by the U.S. Food and Drug Administration for clinical diagnostic use (albeit limited to bacteria and yeasts). In particular, the Bruker Daltonics instrument provides its own solution (i.e., MALDI Biotyper), which is comprised of software and a database, whereas the Vitek MS system integrates the SARAMIS (Spectral Archiving and Microbial Identification System) database (i.e., an open database made by AnagnosTec) with its own closed database. Biotyper software generates (log) score values ranging from 0 to 3, with scores of ≥2 and ≥1.7 being recommended for species-level or genus-level identifications, respectively, whereas a score (confidence) value of ≥60% is recommended for species-level identification using Vitek MS software. Despite differences with regard to sample preparation, spectrum preprocessing, and in silico identification algorithms, all these systems appear to be reliable tools for mold identification.
Results: Over the last 5 years, accumulated experience clearly shows that MALDI-TOF MS holds promise as an accurate mold identification tool, particularly with common filamentous fungal pathogens. Conventional phenotypic methods to identify filamentous fungi are relatively inexpensive but have a turnaround time of several days because of the time taken for fungal growth, which in certain groups of fungi (e.g., dermatophytes) can be very long. As a result, initial antifungal therapy of infection is empirical, and appropriate therapy may be delayed if antifungal resistance is not suspected. The turnaround time has been reduced by the introduction of MALDI-TOF MS instruments in the clinical laboratory routine, although these instruments continue to rely on fungal cultures. In this context, the MALDI-TOF MS technology is also being exploited to analyze patient specimens directly, completely bypassing the need for fungal growth.
Conclusion: The construction of expanded MALDI-TOF MS databases is labor-consuming, requires mycological skills, and is hindered by the fact that the IVD (in vitro device) versions of the commercialized MALDI-TOF MS systems do not enable the implementation of databases. Otherwise, in environments not subject to regulatory-body (i.e., FDA) restrictions, the practice of the expansion/improvement of a RUO (research-use-only) database (i.e., Biotyper or SARAMIS) should be confined to reference laboratories. Thus, while this practice will be a huge benefit for laboratories with large collections of clinical isolates, it should be mandatory that an identification strategy is expanded and validated with new isolates/ species and analyzed with other databases. In this context, efforts are needed to develop online-available reference spectrum databases that could be interrogated similarly to sequence databases such as the NCBI GenBank database.
Keywords: Identification, Dermatophytes, MALDI-TOF MS