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INTRODUCTION: Dermatophytes are a group of fungi which infect keratinized tissues and causes superficial mycoses in humans and animals. The group comprises of three major genera, Trichophyton, Microsporum and Epidermophyton. Among them Trichophyton rubrum is a predominant anthropophilic fungi which causes chronic infections. Although, the infection is superficial and treatable, reinfection/coinfection causes inflation in the treatment cost. Identifying the source and mode of transmission is essential to prevent its transmission. Accurate discrimination is required to understand the clinical (relapse or reinfection) and epidemiological implications of the genetic heterogeneity of this species. Polymorphism in the Non Transcribed Spacer (NTS) region of ribosomal DNA (rDNA) clusters renders an effective way to discriminate strains among T. rubrum. AIM: To carry out the strain typing of the clinical isolates, Trichophyton rubrum using NTS as a molecular marker. MATERIALS AND METHODS: Seventy T.rubrum clinical isolates obtained from April-2011-March 2013, from Sri Ramachandra Medical Centre, Chennai, Tamil Nadu, India, were identified by conventional phenotypic methods and included in this prospective study. The isolates were then subjected to Polymerase Chain Reaction (PCR) targeting two subrepeat elements (SREs), TRS-1 and TRS-2 of the NTS region. RESULTS: Strain-specific polymorphism was observed in both subrepeat loci. Total, nine different strains were obtained on combining both TRS-1 and TRS-2, SREs. CONCLUSION: The outcome has given a strong representation for using NTS region amplification in discriminating the T. rubrum clinical isolates. The method can be adapted as a tool for conducting epidemiology and population based study in T. rubrum infections. This will help in future exploration of the epidemiology of T. rubrum.
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INTRODUCTION: Dermatophytes are keratinophilic fungi causing superficial cutaneous infections that account 20-25% of the global population. As per literature search, there is a dearth in the study on virulence factors of dermatophytes from the Indian sub-continent and moreover the association of the virulence factors and the host tissue in vitro helps in understanding the host-pathogen interaction. AIM: To analyse the enzymatic and non-enzymatic virulence activities of dermatophytes on solid media. MATERIALS AND METHODS: A total of 11 isolates, three standard American Type Culture Collection (ATCC) strains- Trichophyton rubrum- 28188, Trichophyton mentagrophytes- 9533, Trichophyton tonsurans- 28942, one CBS KNAW Fungal Biodiversity Centre strain- Arthroderma grubyi- 243.66, five clinical isolates- T. rubrum, T. mentagrophytes, Trichophyton rubrum var. raubitschekii, Trichophyton interdigitale, Epidermophyton floccosum, and two laboratory isolates - Microsporum gypseum and Microsporum canis were screened for the production of virulence enzymes such as phospholipase, lipase, protease, gelatinase and non-enzyme virulence factors (haemolytic activity) of dermatophytes. The clinical isolates were identified from a tertiary care hospital, Chennai. These dermatophytes were tested upon specific substrates on solid media such as egg yolk, tween 80, bovine serum albumin, gelatin powder and sheep blood respectively. RESULTS: The virulence activity of phospholipase, lipase, protease and gelatinase was observed from all the dermatophyte species. T. rubrum, T. rubrum ATCC strain, T. rubrum var. raubitschekii, T. mentagrophytes, T. mentagrophytes ATCC strain, T. interdigitale and A. grubyi CBS strain produced complete haemolysis, whereas other dermatophytes showed no haemolytic activity. CONCLUSION: Phospholipase, lipase, protease and gelatinase act as enzymatic virulence marker and the T. rubrum complex, T. mentagrophytes complex and A. grubyi showed complete haemolysis and hence they may also act as a non-enzymatic virulence marker for dermatophytes.
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PURPOSE: A pan fungal primer targeting the Internal Transcribed Spacer (ITS) region and optimization of PCR-RFLP using a dermatophyte specific primer targeted the 18S ribosomal DNA (rDNA) region were performed for the identification of dermatophyte species and strains directly from clinical specimens. MATERIALS AND METHODS: One hundred and thirty eight specimens (129 skin scrapings and 9 nail clippings) from clinically suspected cases of dermatophytosis were collected and subjected to direct microscopy and culture. Among them, 66 skin scrapings and 3 nail clippings were processed for genotyping by PCR-RFLP analysis using the Mva I, Hae III and the Dde I restriction enzymes. RESULTS: Of the 138 specimens, 81 specimens were positive for dermatophytosis, the most common one being Trichophyton rubrum (47), followed by Trichophyton mentagrophytes (25) and Epidermophyton floccosum (9). Of the 47 T. rubrum isolates, 10 were T. rubrum var. raubitschekii which were identified phenotypically as urease positive and by DNA sequencing. Since they exhibited minor morphological and physiological features, they have currently been synonymized with T. rubrum. Of the 25 T. mentagrophytes isolates, three were Trichophyton interdigitale, which were identified by DNA sequencing. Among the 66 skin specimens smear, culture and PCR showed the presence of dermatophytes in 36 (54.54%), 42 (63.63%) and 47 (71.21%) cases respectively. Among the three nail specimens, only one was found to be positive for dermatophytosis by smear, culture and PCR. CONCLUSION: Amplification of the dermatophyte specific primer is appropriate in the identification of dermatophytes directly from the clinical material. PCR targeting the ITS region by using the Mva I and the Dde I enzymes was equally good for the RFLP analysis. However, by using the above three restriction enzymes, no strain variations were detected among the T. rubrum and the T. mentagrophytes strains.