Identification Of Dermatophyte Species Research Articles

Report of terbinafine resistant Trichophyton spp. in Italy: Clinical presentations, molecular identification, antifungal susceptibility testing and mutations in the squalene epoxidase gene.

Numerous reports of resistance to terbinafine in Trichophyton spp. from all over the world are arousing justified attention and concern. Point mutations in the gene that encodes the squalene epoxidase (SQLE) enzyme are responsible for these therapeutic resistances. Primary objective of the study was to describe first isolates of Trichophyton spp. resistant to terbinafine among the patients treated between September 2019 and June 2022 at the Dermatology Units of Ospedale Maggiore Policlinico and San Bortolo Hospital. Secondary objective was to study the resistance mechanism. Patients with confirmed Trichophyton spp. infection has been treated with systemic and topical terbinafine. Patients were then re-evaluated 12 weeks after the therapy. Patients with incomplete or absent response to terbinafine underwent a new skin scraping for direct mycological examination, new identification of dermatophyte species from culture and MALDI-TOF, molecular species identification, antifungal susceptibility testing and molecular analysis of SQLE gene. We identified five patients without clinical response to treatment with terbinafine. The DNA sequencing of the ITS region identified one Trichophyton rubrum and four Trichophyton indotineae. The T. rubrum strain showed minimum inhibitory concentration (MIC) (90% growth inhibition) of 4 mg/L for terbinafine. The four T. indotineae strains showed a MICs range of 0.25-4 mg/L for terbinafine. The analysis of the SQLE gene in the T. rubrum strain showed a nucleotide substitution generating a missense mutation (L393F). The SQLE gene sequencing in the T. indotineae strains showed a nucleotide substitution generating a missense mutation (F397L) in two strains, a nucleotide substitution L393S in one strain and a nucleotide substitution F415C in another strain. We report the first cases of terbinafine-resistant Trichophyton isolates in the Italian population. Solid antifungal management programs will be needed to promote more responsible use of antimycotics and preserve their therapeutic efficacy to control antifungal resistance.

Molecular detection and species identification of dermatophytes by SYBR-Green real-time PCR in house methodology using hair samples obtained from dogs and cats.

The classical dermatophytes diagnosis is based on mycological culture and microscopy observation both human and animal hair, skin and nail samples. The aim of this work was to develop the new in-house real-time PCR with pan-dematophyte reaction for detection and identification of the main dermatophytes directly from hair samples, providing a simple and rapid diagnosis of dermatophytosis in dogs and cats. An in house SYBR-Green real-time PCR was designed and used for detecting a DNA fragment encoding chitin synthase 1 (CHS1). A total of 287 samples were processed by culture, microscopic examination with KOH 10% and quantitative PCR (qPCR) analysis. Melting curve analysis of CHS1 fragment revealed to be reproducible, showing a single distinct peak for each species of dermatophyte, namely Trichophyton mentagrophytes, Trichophyton verrucosum Microsporum canis and Nannizzia gypsea (formerly Microsporum gypseum). Then, out of the 287 clinically suspected cases of dermatophytosis, 50% were positive for dermatophytes by qPCR, 44% by mycological culture and 25% by microscopic examination. M. canis was identified in 117 samples tested by culture and 134 samples tested by qPCR, followed by N. gypsea in 5 samples (either tested by culture or qPCR) and T. mentagrophytes detected in 4 and 5 samples when tested by culture or qPCR, respectively. Overall, qPCR allowed the diagnosis of dermatophytosis in clinical samples. The results suggest this newly proposed in house real-time PCR assay can be used as alternative diagnosis and rapid identification of dermatophytes frequently associated to clinical hair samples of dogs and cats.

Identification of Dermatophyte Species by PCR Restriction Fragment Length Polymorphism in Ramallah region, Palestine

Dermatophytes are a group of fungi that cause an infection called dermatophytosis. They are mainly classified into three anamorphic genera: Trichphyton, Microsporum and Epidermophyton. Sixty-five samples were collected during the period from the middle of January to the end of September of the year 2019. The clinical specimens (hair, nail and skin) were collected from 58 patients who were clinically diagnosed with dermatophytosis. Molecular identification was done by using the fungus-specific universal primers (ITS1 and ITS4) for the amplification of the ITS region in the rRNA gene. For species identification, BstN1 restriction enzyme was used for the digestion of the amplified ITS products to produce distinct band patterns. The distribution of dermatophytosis among the patients was 52.3.%, 41.5%, 3.1%, 1.5% and 1.5% for Tinea nail, Tinea capitis, Tinea corporis, Tinea pedis and Tinea imbricate, respectively. Nighnteen clinical isolates of dermatophytes showed positive culture. Trichophyton was the most common genus with 15 sample (78.9%), followed by Microsporum with 4 samples (21.1%) and no Epidermophyton were found. The most common species was T. rubrum (n=5, 26.3%), followed by T. verrucosum (n=3, 15.8%), then T. mentagrophytes, M. Canis, M. audouinii and T. schoenleinii with tow isolates for each (10.5%). The amplification of ITS regions was successful in certain samples by using the fungus-specific universal primers (ITS1 and ITS4) and PCR-RFLP technique

Molecular Methods For Diagnostics Of Dermatomycoses – Review Of Available Techniques And Evaluation Of Their Advantages And Disadvantages In Implementation For In Routine Use

Abstract Fungal infections of the skin, hairs, and nails undeniably dominate among all types of fungal infections. The etiological factors of the majority of superficial fungal infections are dermatophytes which, although they are the oldest microorganisms considered as pathogens, have long been unstable in the taxonomic position. From a diagnostic point of view, the species identification of dermatophytes is still a serious problem, often generating therapeutic errors. An increasing number of infections, including zoonoses, lack of taxonomic stability and ambiguous clinical picture of all cases of dermatomycosis induce to search for new, fast, repeatable and at the same time cheap methods of species identification of these fungi. In the last decade, revolutionary progress has been observed in the development of molecular methods for the diagnosis of fungal infections and the reliable identification of species of etiological factors that cause these dermatomycoses. The results of many studies indicate that the direct identification of fungi from dermatological samples based on molecular methods is much more reliable and much faster compared to that carried out by conventional methods. Often, the etiological factor of the observed changes was also identified, while the result of cultivation was negative. Particular molecular methods used in the species identification of fungi directly from the clinical material differ in the procedures of genomic DNA extraction, PCR techniques used, the molecular marker used and the results interpretation system. This paper reviews literature regarding different methods of diagnosing of superficial fungal infections based on molecular biology techniques, their advantages and limitations, as well as critical factors for their implementation for routine use. The position of microbiologists in this matter seems to be a foregone conclusion, the time when molecular diagnostics will replace the conventional techniques, based on the cultivation of dermatophytes and assessing their morphology, inexorably coming. Molecular methods of identifying aetiological factors of dermatomycoses directly from dermatological samples are much more attractive and have many advantages. 1. Introduction. 2. Importance of identification of dermatophyte species in dermatological samples. 3. Molecular species identification in pure dermatophyte cultures. 4. Methods for direct identification of fungi from clinical samples. 4.1. DNA isolation. 4.2. Classical PCRbased techniques of direct identification. 4.3. Real-time PCR-based techniques of direct identification. 5. Choice of an optimal method for routine use. 6. Advantages and drawbacks of molecular identification methods applied in mycology. 7. Summary

A Survey Of Dermatophytes Isolated From Iraqi Patients In Baghdad City

Dermatophytes infection is a common problem worldwide and frequent in Iraq. Several reports and articles were published on prevalence, distribution, causes and treatment of dermatophytosis .This case study was conducted on fifty patients(31males and 19 females) with suspected dermatophytes were studied . Their ages ranged from one year to fifty years .Patients admitted to Baghdad Teaching Hospital , Dept . of Dermatology , Baghdad during September 2010 to March 2011.Hairs and scales were collected and microscopicall examination using 20% KOH were done. Hair and scales from active outer border of the lesion were inoculated on modified Sabouraud's dextrose agar. Culture was incubated at room temperature(28C°) for 4 - 5 weeks. The identification of dermatophyte species was based on the gross , and microscopical and cultural characteristic according to standard mycological references .The infection of dermatophyte was much higher in children below 10 years of age. Males 31(62%) were affected more than females 19(38%).Tinea capitis 19(47.5%) was the predominant clinical type .The main etiological agents was Trichophyton rubrum 20(50%) followed by Trichophyton mentagrophytes 13(32.5%).The predominant anthropophilic dermatophytic species was Trichophyton rubrum.This study was carried out to determine the prevalence, causative agents of dermatophytosis in group of Iraqi patients in Baghdad.

Clinicomycological study of dermatophytosis in a tertiary care centre

Background: Fungal infections constitute a major health problem all over the world. Signs and symptoms induced by various dermatophytic infections are hardly distinguishable clinically from each other. Hence, characterisation by in vitro culture is required for the appropriate diagnosis and treatment as well as for studying the epidemiological characteristics in a region. Objectives: The objectives of this study are: (1) To isolate and identify dermatophytes affecting skin and nail. (2) To compare two different culture media, namely Sabouraud's dextrose agar (SDA, Himedia Laboratories, Mumbai) with Chloramphenicol and Actidione with dermatophyte test medium (DTM, Hi-Media Laboratories). Materials and Methods: This is a cross-sectional study in which patients attending the outpatient wing of the Department of Dermatology and Venereology, Government Medical College, Thiruvananthapuram, Kerala, India, with clinical features of dermatophytosis were included from March 2011 to February 2012. Skin and nail scrapings were subjected to direct microscopy by 10% potassium hydroxide (KOH), 40% KOH and cultured on SDA with Actidione (Hi-Media Laboratories) and DTM (Hi-Media Laboratories). Results: The total number of samples in this period was 150, of which 99 (66%) samples were positive by direct microscopy and 74 (49.33%) were positive by culture. The most common clinical type was tinea corporis 75 (50%) followed by tinea cruris 40 (26.67%). Out of the 74 isolates, Trichophyton rubrum 40 (54.05%) was the most common species followed by Trichophyton mentagrophytes 29 (39.19%), Microsporum gypseum three (4.05%), Trichophyton schoenleinii one (1.35%) and Epidermophyton floccosum one (1.35%).Nearly 86.1% of the dermatophytes were isolated on DTM within 5-10 days of inoculation whereas 47.05% were isolated on SDA within 10 days of inoculation. This was statistically significant with P < 0.0001 (χ2 = 22.43). Conclusion: DTM can be used as a rapid screening medium for the isolation and identification of dermatophytes compared to SDA with Actidione. However, DTM is inferior to SDA with Actidione in the identification of dermatophyte species.

Clinicomycological Study of Dermatophytosis in South India.

Introduction:Dermatophytic infections are commonly encountered a problem and constitute more than 50% of cases in dermatology outpatient departments. Diagnosis of these infections requires the proper use of laboratory methods.Objectives:This study was conducted to know the etiology of dermatophytosis in patients attending Tertiary Care Level Hospital in South India and to compare the efficacy of Sabouraud's dextrose agar (SDA) with actidione and dermatophyte test medium (DTM) in isolating and identifying dermatophytes.Materials and Methods:A total of 110 samples which included 101 skin samples and 9 hair samples from clinically suspected dermatophytosis were collected. Direct microscopy by KOH and culture on SDA with actidione and DTM were done.Results:Of 110 samples collected, 58.18% were KOH positive for fungal filaments and 56.36% were culture positive for dermatophytes. More number of cases were observed between age groups of 21–40 years. Males were more affected compared to females. Tinea corporis was the common clinical presentation observed (40%). Trichophyton rubrum (58.06%) was the predominant isolate recovered in all clinical presentations but Trichophyton violaceum was the most common isolate in tinea capitis. All culture positives were grown on both SDA with actidione and DTM. Appearance of growth was earlier on DTM that is, within 10 days compared to SDA with actidione where growth started appearing only after 10 days. This is statistically significant P < 0.0001 (χ2 = 71.6). Species level identification on primary isolation was possible when grown on SDA with actidione and it was not possible with the growth on DTM on primary isolation.Conclusion:DTM is a good screening medium in laboratory diagnosis of dermatophytosis when compared to SDA with actidione. But DTM is inferior to SDA with actidione in identification of dermatophyte species.

Updating the epidemiology of dermatophyte infections in Palestine with special reference to concomitant dermatophytosis

To determine the epidemiology of dermatophytosis in Palestinian patients, detect changes in the etiological agents during the last three decades, and to correlate between concomitant tinea pedis infections, and other cutaneous lesions. 220 suspected dermatophytosis patients were involved in this study. In an additional 38 cases, where consultation was prompted by tinea pedis, the presence of other lesions of concomitant dermatophytosis was studied, to further investigate the diagnosis. Clinical specimens were collected and identification of dermatophyte species was based on gross and microscopic morphology. Epidemiology of tinea capitis has gone the most radical changes in Palestine in the last three decades, with the zoophilic dermatophyte Microsporum canis replacing Trichophyton violaceum, becoming the predominant causative agent. During this study, 21.6% (38/176) patients with tinea pedis and concomitant lesions caused by the same dermatophytes at sites distant from the primary lesions in the foot were prospectively identified. About 63.2% of patients with tinea pedis have a concomitant toenail onychomycosis infection. The epidemiology of dermatophytosis, especially tinea capitis, has gone the most radical changes in Palestine in the last three decades, with M.canis replacing T.violaceum, and becoming the predominant causative agent of all cases of infections. The coexistence of tinea pedis with other types of fungal skin infections is a frequent phenomenon; we believe that the infected foot may be a site of primary infection. Thus, the effective therapy for tinea pedis is essential to prevent spreading the infection to other sites of the skin.

Species Identification of Dermatophytes by MALDI-TOF MS

MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) is a new tool for the identification of microorganisms, inclusive of dermatophytes. The technique is faster, more straightforward, and powerful when compared to conventional dermatophyte identification methods (culture and microscopy). Accurate species identification in dermatophytes is not only essential to survey epidemiologic situations, but also for an appropriate medical treatment and to locate the source of infection (zoophilic or anthropophilic). Multiple platforms from a number of well-established commercial manufacturers (Andromas SAS, Bruker Daltonics, bioMerieux) have been used for dermatophyte identification with different success. Independent from the platform used, all of the studies reviewed here report on problems with the identification of phylogenetically closely related anthropophilic and zoophilic dermatophyte species. Thus, supplementation of the databases/libraries as well as standardized extraction protocols and cultivation methods are the precondition required for optimal species identification.

Dermatophytoses in domesticated animals

Dermatophytes are among the most frequent causes of ringworm infections in domesticated animals. They are known to serve as reservoirs of the zoophilic dermatophytes and these infections have important zoonotic implication. In Nigeria and probably West Africa, there are not many studies on the incidence of dermatophytosis in domesticated animals. In the current study, 538 domesticated animals with clinically suggestive lesions were investigated for dermatophytes. Identification of dermatophyte species was performed by macro- and micro morphological examination of colonies and by biochemical methods. In the cases of isolates that had atypical morphology and/or biochemical test results, the rDNA internal transcribed spacer region 2 (ITS 2) sequencing was performed. Out of this number, 214 (39.8%) were found to be colonized by a variety of ten species of dermatophytes. M. canis was the most frequently isolated species (37.4%), followed by T. mentagrophytes (22.9%) and T. verrucosum (15.9%). M. persicolor and T. gallinae were jointly the least species isolated with a frequency of 0.55% respectively. The recovery of dermatophyte isolates previously shown to be common etiological agents of dermatophytosis especially from children in the same region suggests that animal to human transmission may be common. Possible implications and recommendations are discussed.

Changes of Dermatophytoses in Southwestern Greece: An 18-Year Survey

The isolation and distribution rate of dermatophytes as causative agents of superficial mycoses of skin, hair, and nails during an 18-year period (1991-2008) at a university hospital are presented. A comparative analysis of epidemiological differences within the first (1991-1999) and the second 9-year period (2000-2008) was performed. Skin scrapings, nail, and hair specimens were examined by a direct microscopic examination and culture. Identification of dermatophyte species was based on macroscopic and microscopic characteristics of colonies. During the complete period (18 years), 5,971 patients with suspected dermatophytosis were examined. Seven hundred and sixty-nine patients (12.8%) were found positive. Among them, 495 cases (64.3%) were of skin dermatophytoses, 91(11.8%) of hair, and 183 (23.7%) of nails. The most frequent etiological agents were Microsporum canis (54%), Trichophyton rubrum (38%), and T. mentagrophytes (6%). Epidermophyton floccosum, T. tonsurans, T. violaceum, and M. gypseum were responsible only for 16 cases (2%) of dermatophytoses. The prevalence of dermatophytoses seems to decrease significantly from 16.2% (1991-1999)-9.6% during the last 9-year period. The most frequent dermatophyte, M. canis, shows decreasing trends during the last period (from 58.5 to 45.7%), whereas T. rubrum shows an increasing isolation rate (from 35 to 43.6%), respectively. The most common form of dermatophytosis among children remains tinea capitis due to M. canis. The most frequent etiological agent of tinea unguium (81%) is T. rubrum.

Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI‐TOF mass spectrometry

Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI-TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit.

We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex.

Intracellular free amino-acids of certain dermatophytes.

THE identification and classification of the dermatophytes is dependent on macroscopic and microscopic morphological characteristics developed during growth on standard media. The formulation of biochemical tests to assist in identification would be of considerable value. The work of Mattick et al. 1, which showed that extracting cells of various species of Lactobacilli with 10 per cent acetic acid removed a mixture of amino-acids and peptides from the cells which was characteristic for each species and constant for strains within a species, suggested the application of a similar technique for the identification of dermatophyte species.