Low frequency of itraconazole resistance found among Aspergillus fumigatus originating from poultry farms in Southwest Poland.

AIM: The aim of this study was to investigate the collection of avian Aspergillus fumigatus isolates for the presence of triazole resistance. MATERIAL AND METHOD: The study was performed on 60 A. fumigatus isolates cultured from lung tissue samples from chicken (25), geese (17), turkeys (13) and ducks (5). The samples were obtained from 40 different farms located in the Southwest Poland and were collected in the period of September 2015 to November 2016. The EUCAST microdilution method, with the use of three concentrations of itraconazole (ITR) (1, 0.5, and 0.25mg/L), was used to screen the susceptibility of all isolates. Additionally, the selected 20 isolates were tested with eleven concentrations ranging 0.015-16mg/L of ITR, voriconazole, posaconazole and isavuconazole. RESULTS: Most tested isolates (59/60) were susceptible to ITR (MIC≤0.5mg/L). One isolate showed elevated MIC for ITR (16mg/L), as well as voriconazole (4mg/L), izavuconazole (4mg/L), and posaconazole (0.5mg/L). This isolate was identified on the basis of DNA analysis as A. fumigatus carrying TR34/L98H mutation. All of the ITR-susceptible isolates under study were also susceptible to other triazoles. CONCLUSION: Obtained results indicated a low frequency (1.6%) of A. fumigatus resistant to triazoles among avian isolates from the Southwest regions of Poland.

PCR and real-time PCR approaches to the identification of Arthroderma otae species Microsporum canis and Microsporum audouinii/Microsporum ferrugineum.

OBJECTIVES: The identification of species in the Arthroderma otae complex is essential to determine the origin of infection and to eliminate the risk of transmission. Microsporum canis is a zoophilic species, whereas Microsporum audouinii and Microsporum ferrugineum are anthropophilic species. In this paper, we propose alternative methods that permit species-specific identification of both anthropophilic and zoophilic members of the A. otae complex METHODS: Two PCR assays were designed based on differences in the DNA fragment encoding ß-tubulin and were applied in both traditional and real-time PCR using DNA isolated by rapid method from culture. RESULT: The two assays presented in this study enable the identification of M. canis and M. audouinii/M. ferrugineum with 100% sensitivity and specificity by both traditional and real-time PCR. CONCLUSION: We developed a new diagnostic assay using specific primers and both traditional and real-time PCR reactions that can be applied in routine laboratory praxis as well as in epidemiological studies to detect M. canis and M. audouinii/M. ferrugineum DNA from a pure culture.

Experimental guinea pig model of dermatophytosis: a simple and useful tool for the evaluation of new diagnostics and antifungals.

The aim of this study was to establish a simple guinea pig model for the purpose of evaluating diagnostic principles and treatment modalities for dermatophytic infections. The following variables were evaluated; pre-treatment of the skin by shaving versus tape stripping, Microsporum canis or Trichophyton mentagrophytes test strains as etiologic agents, differences in inoculum concentrations, and inoculation with and without occlusion. The course of infection was evaluated clinically by redness and lesion scores and mycologically by microscopy, culture, and histopathology. The applicability of the model was evaluated with a recently developed diagnostic pan-dermatophyte PCR and antifungal treatment was tested with an oral solution of itraconazole, 10 mg/kg, once daily during days 3-14 of the test period. Pre-treatment of the skin with a manual razor was for practical reasons preferable to tape stripping. Inoculation under occlusion showed no advantage in the establishment of experimental infections. Infection severity showed some association with the inoculum concentration and subtype of T. mentagrophytes but not in studies involving M. canis. The establishment of dermatophytosis was confirmed by histopathology. Surprisingly, microscopy was found to be less sensitive than culture and the latter was as sensitive as pan-dermatophyte PCR. Itraconazole significantly reduced lesion and redness score, with M. canis infections responding better to itraconazole treatment than those caused by T. mentagrophytes. In conclusion, we established a dermatophytosis animal model, which was proven useful for evaluating diagnostic methods and antifungal susceptibility testing.

Fluorescent protein vector for directional selection of PCR clones.

Green fluorescent protein (GFP) has become a convenient and versatile tool as a reporter protein in many aspects of science. Here, we show that the enhanced yellow fluorescent protein (EYFP) variant may be used advantageously as a reporter system for directional cloning of blunt-ended PCR products. We have constructed a pUC18-derived plasmid containing a reporter gene coding EYFP cloned into the BamHI/HindIII sites. The blunt-ended PCR product is cloned into the SmaI site of that plasmid. A reverse PCR primer must be designed with extra bases on the 5' end that are required to introduce a ribosome binding site (rbs) for EYFP expression. The reporter gene coding EYFP is not expressed unless an rbs is introduced in the proper orientation at the 3' end of the cloned PCR insert. The results of this cloning procedure may be analyzed by simple visual inspection using a transilluminator. In most cases, successful directional cloning results in white fluorescent colonies. The proposed procedure is a convenient method that can reduce the time- and labor-intensive analysis of the clones obtained during blunt-ended PCR product cloning.