Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae.

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains.

Evaluation of a Liquid Media MALDI-TOF MS Protocol for the Identification of Dermatophytes Isolated from Tinea capitis Infections.

One of the most common types of tinea is the superficial infection of the hair and scalp area known as tinea capitis. It is responsible for frequent outbreaks in nurseries and schools and represents a global health problem. Correct identification of the infection agent is essential in the determination of the infection source, epidemiological course, and treatment initiation. The conventional identification methods (direct exam, culture, DNA sequencing) are time-consuming, require experienced staff, are time-consuming, and the latter is expensive for routine identifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is gaining new ground for routine identification of filamentous fungi. The main advantages of MALDI-TOF MS are its rapid and accurate identification capability, relatively low cost, and easy integration into the laboratory routine. Its accuracy heavily depends on the quality of the reference spectra database. Identification of clinical isolates with MALDI-TOF MS protocol requires a sub-culturing step to ensure reliable identification. It can take days to weeks before fungal growth appears on solid medium. In this study, a unique MALDI-TOF MS protocol using liquid cultures of dermatophyte species was developed in order to shorten the turnaround time for the culture and identification of clinical isolates. Material and Method A standard MALDI-TOF MS protocol was adapted for liquid instead of solid cultures. Three different databases were tested. Results Using the liquid media MALDI-TOF MS protocol, a global rate of 62% correct identification (RCI) was obtained, compared with 87% for the protocol based on solid cultures. Trichophyton tonsurans was not correctly identified in all isolates using liquid cultures, with 88% of the isolates misidentified as Trichophyton interdigitale. The turnaround time for primary isolates for the solid and liquid protocols were respectively 11.7 and 11.6 days (no significant difference between both methods (p = 0.96)). Conclusions The newly designed liquid MALDI-TOF MS protocol did not lead to a significantly shorter turnaround time for the identification of dermatophytes isolated from tinea capitis infections. The turnaround time for the method with primary isolates was not significantly lower, and the rate of correct identification decreased remarkably, which emphasizes the need for a sub-culturing step. Using different database did not lead to improvement in turnaround time or rate of correct identification. This study highlights the importance of the medium and the reference database when performing MALDI-TOF MS.