Anita Hryncewicz-Gwóźdź

Identification and differentiation of Trichophyton rubrum clinical isolates using PCR-RFLP and RAPD methods

Trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. Several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. The purpose of this study was to apply selected DNA fingerprinting methods to the identification and strain discrimination of T. rubrum clinical isolates. Fifty-seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and strain differentiation using a randomly amplified polymorphic DNA (RAPD) method, with two primers designated 1 and 6. Using PCR-RFLP, 55 of the isolates studied were confirmed to be T. rubrum. Among those, a total of 40 and five distinct profiles were obtained by RAPD with primers 1 and 6, respectively. The combination of profiles from both RAPD assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. A dendrogram analysis performed on the profiles generated by RAPD with primer 1 showed most of the isolates (87.3%) to be genetically related. PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum species, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing.

Molecular typing of Trichophyton rubrum clinical isolates from Poland

The aim of this study was to investigate the intraspecific diversity of Trichophyton rubrum clinical isolates. Thirty clinical isolates of T. rubrum were selected for molecular typing by PCR amplification of two tandemly repetitive elements (TRS‐1 and TRS‐2) of the rDNA and randomly amplified polymorphic DNA (RAPD) analysis with primers designated 1 and 6. The assignment to the species T. rubrum was achieved by nested PCR of ITS1. Five PCR types were produced from the TRS‐1 and three from the TRS‐2 locus. Thirteen and 23 individual profiles were obtained by RAPD, with primer 1 and 6 respectively. At the phylogenetic level, 26 (87%) isolates were allocated into four clusters, with each cluster comprising isolates of over 80% similarity. The reproducibility of TRS typing was 100%, whereas that of RAPD was 40% and 30%, when using primer 1 and 6 respectively. Neither correlation between the morphological characteristics and the TRS‐1‐TRS‐2 or RAPD genotype nor between TRS‐1‐TRS‐2 and RAPD genotyping was observed. Although both the TRS amplification and RAPD analysis possess the ability to discriminate between T. rubrum strains, the TRS typing method is particularly valuable as its results are much more reproducible, more easily interpreted and recorded than those generated by RAPD.

Tinea capitis and tinea corporis with a severe inflammatory response due to Trichophyton tonsurans.

Trichophyton tonsurans is an anthropophilic dermatophyte, with a worldwide distribution, although its prevalence varies considerably between different geographical regions. Whereas in North America infections due to this fungus are exceptionally common, on the European continent they appear relatively seldom. Although T. tonsurans is primarily associated with tinea capitis, it can also be the cause of tinea corporis and tinea unguium. The course of infection is usually only mildly symptomatic. We describe here two cases of urease-positive T. tonsurans infections with atypically extensive cutaneous lesions and severe inflammatory responses. .

Detection and identification of human fungal pathogens using surface-enhanced Raman spectroscopy and principal component analysis

This paper demonstrates that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast and reliable technique for the detection and identification of human fungal pathogens, such as Trichophyton rubrum, Candida krusei, Scopulariopsis brumptii, and Aspergillus flavus. Fungal infections have become one of the leading infectious causes of morbidity and mortality among hospitalized patients and/or immunocompromised hosts. Hence, there is a strong need for the development of new technologies allowing for fast and reliable diagnosis of fungal diseases. Our study shows that the SERS technique effectively distinguishes between selected common fungal pathogens and thus offers taxonomic affiliation of fungi within several minutes. Additionally, the PCA analysis allows performing statistical classification of fungal pathogens studied and identifying the fungal spectrum directly from a clinical sample. Calculated two principal components (PCs) (PC-1, PC-2) are the most diagnostically significant, explain 97% of the variability and enable, with very high probability, discrimination between the four mentioned fungal species. Moreover, the results of this study demonstrate the excellent possibility for the identification of fungi from human skin samples. The research presented in this paper offers an alternative for conventional fungal diagnostics and paves the way for the development of a new, fast, robust, and cost-effective diagnostic test for the detection and identification of fungal pathogens.

Stability of Tandemly Repetitive Subelement PCR Patterns in Trichophyton rubrum over Serial Passaging and with Respect to Drug Pressure

Trichophyton rubrum is the most significant agent of dermatomycoses worldwide, primarily causing tinea pedis and tinea unguium. PCR analysis of tandemly repetitive subelements (TRS) within the rDNA nontranscribed spacer region is a major tool for molecular typing of T. rubrum. The aim of this study was to investigate the stability of TRS PCR patterns by analyzing isogenic strains of T. rubrum. Twenty-seven groups of isogenic T. rubrum strains were examined, each composed of an original clinical isolate and its 3 subcultures, maintained on a drug-free medium, a medium containing fluconazole and itraconazole. TRS typing was performed for the original strains and their subcultures grown after 12 passages, at 4-week intervals, on respective media. To add more objectivity to the results, TRS typing for each of the isogenic strain was performed three times, using DNA isolated from three different colonies. Among 27 groups of isogenic strains, all but one were exclusively composed of strains with identical TRS-1 and TRS-2 PCR patterns. In one group, 3 isolates from the last, twelfth passage had identical TRS-1 PCR profiles (type 1), yet different TRS-2 PCR profiles, as compared with the original strain (type I vs. type II). The mechanism underlying the genotype switch was a deletion of a single repeat unit in the TRS-2 locus, as evidenced by sequence analysis. In the interpretation of TRS typing results, microevolutionary events need to be taken into account, urging drawing epidemiological conclusions with caution and in conjunction with other genotyping data and traditional contact tracing information.

Multiple disseminated keratoacanthoma-like nodules: a rare form of distant metastases to the skin

Cutaneous metastases are found in approximately 0.7–10.4% of internal malignancies and they may rarely be the first symptom of the underlying neoplasm [1]. Typically, cutaneous secondaries present as a single, erythematous nodule, occasionally ulcerated, however, other presentations, including erysipelas carcinomatosa, alopecia neoplastica or carcinoma en cuirasse in the course of the breast cancer, or angiomatous tumors in the course of renal carcinoma may be occasionally observed. The metastases assimilating keratoacanthomas are extremely rare [2, 3]. Herein, we present a 72-year-old man, cigarette smoker, who was referred to our department with disseminated skin tumors of unknown etiology. On admission, domeshaped, inflamed tumors, some of them with central, keratin-filled craters, clinically mimicking keratoacanthomas were observed on the scalp, forehead, nose, neck and trunk (back and left shoulder) (Figures 1, 2). All the

Diffuse melanosis cutis related to dermal micrometastases as the first clinical symptom of distant metastatic malignant melanoma: Case report

Rationale: Diffuse melanosis cutis (DMC) is a very rare sign of malignant melanoma progression. The condition usually develops after approximately one year from melanoma diagnosis in a patient with metastatic tumors and after anticancer treatment with cytostatic medications. Patient concerns: A 72-year old Caucasian man was admitted to the Department of Dermatology with DMC for 4 months and the history of two melanomas treated surgically 30 years and 9 months before present hospitalization. Diagnosis: Histological and immunohistochemical examinations of DMC biopsy indicated melanoma metastatic cells as well as free deposits of melanin and melanophage presence in the dermis. Interventions: The patient refused to the treatment. Outcomes: The patient died eight months after DMC appeared. Lessons: DMC is a rare presentation of advanced MM and is a bad prognostic factor. The pathomechanisms of the discoloration of the skin are not fully explained. The role of micrometastases, as well as melanin precursors, released during lysis of MM metastases, and growth factors may play a role in the development of the symptom.

Paradoxical Reaction During a Course of Terbinafine Treatment of Trichophyton interdigitale Infection in a Child.

Report of a Case | A child weighing 30 kg and without any comorbidities and allergies and not taking any long-term medications was seen for erythematous, infiltrating, welldemarcated skin lesions located on the face, chest, abdomen, and limbs (Figure, A). Mycological culture yielded growth of Trichophyton mentagrophytes. The fungus was identified as T interdigitale by sequence analysis of the internal transcribed spacer and the D1/D2 domains of the large-subunit (26S) rRNA gene within the rDNA cluster using the polymerase chain reaction sequencing described elsewhere.1 The patient reported frequent contact with stray cats. Initial treatment included 125 mg of terbinafine once daily. Twelve hours after the first dose, we observed temperature elevation (39°C), chills, malaise, aggravation of inflammatory symptoms including increased erythema, edema, and pustule formation (Figure, B). These changes were accompanied by increased erythrocyte sedimentation rate (47 mm/h), elevated C-reactive protein levels (25.8 mg/L), leukocytosis (white blood cell count, 15 390/uL), eosinophilia (eosinophils, 11%), and high IgE level (7200 μg/L). (To convert C-reactive protein to nanomoles per liter, multiply by 9.524; white blood cells to ×109/L, multiply by 0.001; and IgE to milligrams per liter, multiply by 0.001.) Therapy with terbinafine was discontinued for 3 days and oral prednisone was administered (10 mg/d). Terbinafine therapy was reintroduced at the same dose (125 mg/d) for 6 weeks resulting in a complete resolution of skin lesions.

Extensive chronic Tuberculosis luposa treated incorrectly with long-term course of isoniazid monotherapy.

© 2013 The Authors. doi: 10.2340/00015555-1419 Journal Compilation