New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.

Cold atmospheric pressure plasma (CAPP) as a new alternative treatment method for onychomycosis caused by Trichophyton verrucosum: in vitro studies

Purpose Anthropophilic dermatophytes as etiological factors of onychomycoses are more common than zoophilic fungi. In the case of the latter, reverse zoonoses are possible, which poses a threat to the persistence of dermatophytes in the environment. Nevertheless, without treatment, both types of tinea unguium may lead to complete nail plate destruction and secondary mixed infections with fungi and bacteria. One of the zoophilic dermatophytes that cause onychomycosis is Trichophyton verrucosum, whose prevalence has been increasing in recent years. Such infections are usually treated with allylamines and/or azoles, but such a conventional treatment of infections caused by T. verrucosum often fails or is discontinued by patients. Methods Herein, we reveal the results of our in vitro studies related to direct application of cold atmospheric pressure plasma (CAPP) on Trichophyton verrucosum growth, germination and adherence to nail as a new alternative treatment method of such types of dermatomycoses. Results Our in vitro studies showed that, while exposure to CAPP for 10 min delays germination of conidia and clearly impairs the fitness of the fungal structures, 15 min is enough to kill all fungal elements exposed to plasma. Moreover, the SEM images revealed that T. verrucosum cultures exposed to CAPP for 10 and 15 min were not able to invade the nail fragments. Conclusion The results revealed that single exposure to CAPP was able to inhibit T. verrucosum growth and infection capacity. Hence, cold atmospheric pressure plasma should be considered as a promising alternative treatment of onychomycoses.

Trichophyton Species Isolated from Asymptomatic Patients of the Pet-owner Pair in Mexico

Background and Purpose: Superficial mycoses are the fourth most common cause of disease worldwide. It is not surprising that zoonotic transmission occurs to humans due to close contact with different animals, be it companion or farm animals. Therefore, the objective of this study was to determine the presence of asymptomatic dermatophyte carriers in the owner-pet pairs, identify the most common etiologic agents, and find the likely connection between the carrier status of an owner and the presence of dermatophytes in their pets. Materials and Methods: From May 2019 to January 2020, 21 cats and 115 dogs with their respective owners were selected for dermatophyte culture. All the dogs and cats included in the study were from the communities of southeastern Mexico. The samples were taken with a cotton swab, which was vigorously rubbed and twisted on the scalp or body of the pet four times and grown on Mycosel Agar. The isolates were identified based on macroscopic and microscopic characteristics. The prevalence of the binomial ranged from 0.73% in pet skin and human hands to 2.2% in human scalp. In humans, the agents were Trichophyton tonsurans and Trichophyton verrucosum, while in pets, a strain of Trichophyton sp was found. Conclusion: Different species of dermatophytes were found in the owner/pet pairs, which denotes that coexistence is not related in asymptomatic cases.

Modulation of ERG gene expression in fluconazole-resistant human and animal isolates of Trichophyton verrucosum

Dermatophytes are a group of eukaryotic microorganisms characterized by high capacity to colonize keratinized structures such as the skin, hair, and nails. Over the past years, the incidence of infections caused by zoophilic species, e.g., Trichophyton verrucosum, has been increasing in some parts of the world, especially in Europe. Moreover, the emergence of recalcitrant dermatophytoses and in vitro resistant dermatophytes has become a cause of concern worldwide. Here, we analyzed the mechanisms underlying resistance to fluconazole among clinical isolates of T. verrucosum. Quantitative RT-PCR was carried out to determine the relative expression levels of mRNA transcripts of ERG3, ERG6, and ERG11 genes in the fungal samples using the housekeeping gene GAPDH as a reference. Our results showed that the upregulation of the ERG gene expression is a possible mechanism of resistance to fluconazole in this species. Furthermore, ERG11 is the most statistically significantly overexpressed gene in the pool of fluconazole-resistant T. verrucosum isolates. Additionally, we have demonstrated that exposure to fluconazole increases the levels of expression of ERG genes in fluconazole-resistant isolates of T. verrucosum. In conclusion, this study has shown one of the possible mechanisms of resistance to fluconazole among zoophilic dermatophytes, which involves the maintenance of high levels of expression of ERG genes after drug exposure.

SKIN DISEASES OF DIFFERENT ETIOLOGY IN DOGS

Currently, skin pathologies in animals are of close attention by veterinary practitioners, since the qualities of service dogs are reduced against their background, as well as natural resistance is decreased, which contributes to the emergence of skin diseases of different etiology. In this regard, a goal was set to study and analyze the occurrence of skin diseases of different etiology in dogs. To study skin pathologies in dogs, animals were examined from 2010–2018. According to the results of the data obtained, we found that skin diseases were widespread. Among skin pathologies, diseases of parasitic etiology, allergic diseases, infectious skin diseases, diseases caused by formation of neoplasms, as well as endocrine dermopathy were recorded. At the same time, the taxonomic composition of parasites in dogs was represented by 8 species, of which the class Arachnida, Cuvier, 1812 by five species of parasites, and the class Insécta, Linnaeus, 1758 by three representatives. For skin pathologies of infectious nature, representatives of these classes are Bacillales Erenberg, 1835 (Staphylococcus Rosenbach, 1884; Streptococcus Rosenbach, 1884) and the class Eurotiomycetes Microsporum Gruby 1843; Trichophyton verrucosum (Boden, 1902).