Identificación de aislamientos clínicos del complejo Sporothrix schenckii por espectrometría de masas MALDI-TOF

[Extracto] Introducción: Sporothrix schenckii es un hongo dimórfico, que por mucho tiempo fue considerado la única especie responsable de la esporotricosis. Sin embargo, con el advenimiento de las técnicas de biología molecular, aplicadas a la investigación taxonómica de hongos, se demostró que S. schenckii es un complejo de especies crípticas conformado por S. brasiliensis, S. schenckii sensu stricto, S. globosa, S. mexicana y S. luriei. Estas especies difieren en sensibilidad antifúngica, distribución geográfica y virulencia. Tradicionalmente la identificación de Sporothrix spp., se ha llevado a cabo con métodos fenotípicos clásicos, sin embargo, estos no son suficientes para la clasificación correcta de las diferentes especies, por la similitud morfológica entre ellas. La espectrometría de masas (EM) MALDI-TOF es una técnica ampliamente utilizada para la identificación rápida de diferentes microorganismos. El objetivo del presente trabajo fue la creación y validación de una base de espectros de diferentes especies del complejo S. schenckii a partir de la forma micelial del hongo para identificar aislamientos clínicos por EM- MALDI-TOF. [...]info:eu-repo/semantics/publishedVersio

Identificación por secuenciación y por espectrometría de masas MALDI-TOF y evaluación de la susceptibilidad antifúngica in vitro de aislamientos clínicos de Neoscytalidium spp.

[Extracto] Introducción: Neoscytalidium spp. son hongos dematiáceos, queratinofílicos que habitan en suelo y plantas. La taxonomía de este género ha sido problemática y constantemente revisada. Actualmente se reconocen las especies N. novaehollandiae, N. orchidacearum y N. dimidiatum, ampliamente distribuidos en regiones tropicales y subtropicales. Aunque estas especies son generalmente fitopatógenas, estudios han demostrado la implicación de N. dimidiatum en infecciones de piel, uñas, tejido celular subcutáneo y extracutáneo. La identificación de estos hongos se ha basado en la observación de las características macroscópicas y microscópicas de los cultivos in vitro, lo que puede conducir a errores. Como alternativa se puede considerar la secuenciación y la espectrometría de masas (EM) MALDI-TOF; esta última se utiliza con éxito en la identificación de bacterias y levaduras debido a la rapidez y costo-efectividad. Sin embargo, en la identificación de hongos filamentosos aún presenta inconvenientes. Por otro lado, N. dimidiatum es poco sensible a diferentes antifúngicos de uso clínico. Considerando lo anterior, el objetivo de este estudio fue identificar aislamientos clínicos de Neoscytalidium spp. por secuenciación y por EM MALDI-TOF y evaluar la susceptibilidad in vitro a tres antifúngicos. [...]info:eu-repo/semantics/publishedVersio

Identification of dermatophytosis and onychomycosis etiologic agents by classic and by MALDI-TOF MS phenotypic methods

Skin, nail and hair infections are a frequent cause of dermatological consultation in tropical countries. The fungi involved in these infections are usually dermatophytes, Candida spp and some non-dermatophyte filamentous fungi (NDFF). Within the NDFF group, Neoscytalidium dimidiatum has been increase as common agent of onychomycosis. The identification of these etiologic agents, in routine mycological diagnosis, is mainly based on macro- and micro-morphology characters. However, this approach is subjective and the identification could be not accurate. This is an obstacle for the implementation of appropriate therapy, since that antifungal susceptibility profiles may vary among species. Currently, more sensitive, specific, rapid and cost-effective techniques are being introduced for the identification of microorganisms, such as Matrix Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry (MS) with the protein profile of each microorganism. The aim of our study was to compare the identification by classic and MALDI-TOF MS phenotypic methods fungal isolates collected from patients with a clinical diagnosis of onychomycosis or dermatophytosis. In total, 134 clinical isolates were included in the study and identified by classic macro- and micro-morphology characters from de cultures. In addition, the isolates were analysed by MALDI-TOF MS. An in house spectra library was establish in order to identify N. dimidiatum, because this species is not included in commercial libraries. Identification by classic approach resulted in 76 dermatophytes with T. rubrum (41), T. mentagrophytes complex (15), T. tonsurans (6), E. flocossum (2), M. gypseum (1) and M. canis (11); 26 Candida with C. albicans and C. tropicalis representing 5 and 4 isolates, respectively; for NDFF the prevalent species was N. dimidiatum with 25 out of 31 isolates, follow Fusarium spp (4), Aspergilllus flavus (1) and Penicillium sp (1). One Trichosporon sp was also identified. For MALDI-TOF MS the dermatophytes isolates identified as T. rubrum and M. canis reduced to 16 and 4, respectively and the prevalent group became Trichophyton spp (37). For NDFF, MALDI-TOF MS confirmed the identification at species level of 19 N. dimidiatum, the isolate A. flavus and at genus level the 4 Fusarium spp. Within the NDFF isolates, the Penicillium sp isolate was correctly identified by MALDI-TOF MS as A. versicolor. The Trichosporon sp isolate was also confirmed. Finally, the identification of Candida isolates by MALDI-TOF MS were resolved at species level with C. parapsilosis (6), C. haemulonii (1) and C. guilliermondii (1). The inconsistencies observed in the results from the two approaches used with more evidence for filamentous fungi than for yeasts identifications can be due to: 1) the established score to define the identification up to genus or species level; 2), sample preparation and the quality of the extraction of proteins; and, 3) the lack of spectra or low number of species included in the commercial libraries could interfere in the final outputs. The results of this work clearly show that the classic approach is prone of errors, much more work is needed to make MALDI-TOF MS identification a routine in clinical laboratories and to clarify these results identification by molecular biology is advised.info:eu-repo/semantics/publishedVersio

ITS rDNA gene analysis versus MALDI-TOF MS for identification of Neoscytalidium dimidiatum isolated from Onychomycosis and Dermatomycosis cases in Medellin (Colombia)

Within the Neoscytalidium genus, N. dimidiatum, N. oculus, N. orchidacearum, and N. novaehollandiae have been recognized. Although these species are frequently found in soil, N. dimidiatum has been identified as an etiologic agent of onychomycosis or dermatomycosis, and N. oculus has been identified as an etiologic agent of an ocular lesion. All these species can be cultured in vitro, but their morphological identification by macroscopic and microscopic traits is difficult and imprecise due to their similarity. In this study, 34 isolates of Neoscytalidium spp. from 32 onychomycosis and two dermatomycosis cases in Medellin (Colombia) were identified at the species level using sequencing of the ITS1+5.8S+ITS2 nuclear rDNA region and MALDI-TOF mass spectrometry (MS). Neoscytalidium dimidiatum strain MUM 17.21 was used to construct the reference spectrum in the in-house library to identify the clinical isolates by MALDI-TOF MS. Additionally, N. dimidiatum PPC-216 and PLAB-055 strains were used to validate the in-house constructed reference spectra. Although four groups were observed in the dendrogram obtained from the proteins of each isolate profile, MALDI-TOF MS and sequencing results are in accordance, since all isolates were identified as N. dimidiatum.Financial support for this work was provided by the Comité para el Desarrollo de la Investigación, Universidad de Antioquia, Medellín, Colombia. Grant No 2604 and also partially supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Molecular and proteomic identification of Neoscytalidium dimidiatum as onychomycosis and dermatomycosis etiologic agent

[Excerpt] The Botryosphaeriaceae family includes genera frequently related with plant diseases. Nevertheless, human infections caused by fungal plant pathogens are becoming more frequent. Neoscytalidium species are mainly phytopathogenic but cases of phaeohyphomycosis have been reported. Within this genus. four species are currently recognized: N. dimidiatum, N. orchidacearum, N. novaehollandiae, and N. oculus. Although N. dimidiatum is frequently found in soil. it has been identified as an etiologic agent of onychomycosis or dermatomycosis. On the other hand, N. oculus was isolated and identified as an etiologic agent of an ocular lesion. All these species are very similar with regard to their macroscopic and microscopic traits, which lead to complex and imprecise identification based on morphological traits alone. [...