ELUCIDATING THE DERMATOPHYTE SPECTRUM THROUGH RAPID MULTIPLEX PCR AND DETECTION OF ANTIFUNGAL DRUG RESISTANCE CAUSED BY MUTATIONS IN SQUALENE EPOXIDASE AND 14-ALFA DEMETHYLASE GENE
Trial Snapshot
- Phase
- Not Applicable
- Status
- Recruiting
- Sponsor
- IADVL
- Enrollment
- 100
- Locations
- 1
- Primary Endpoint
- Identification of species of dermatopytes and drug resistance in dermatophytic skin infection by rapid methods1
Study Overview
Brief Summary
All clinically identified cases of dermatophyte skin infections not on treatment with topical and/or systemic antifungal from 2 weeks prior will be included in the study. Skin scrapings will be collected using no.15 sterile blade on the Dermatophyte Test Media and KOH mount will be done. The media that shows growth of filamentous fungi will be then subcultured on to SDA for species identification.
A portion of the skin scrapings (~100 mg) will be aliquoted for DNA extraction. Fungal DNA will be extracted from the skin scraping . The extracted DNA will be quantified using a
Nanodrop UV spectrophotometer. Conventional PCR and Species specific RT PCR will be performed . It is performed for live fungi where protein synthesis is taking place. PCR products will be resolved by horizontal gel electrophoresis and results will be analyzed using the gel documentation system. The amplicons will be purified and then sequenced. Sequences will be analyzed using bioinformatics tools to identify
species and determine the clonal nature of the fungi detected. Squalene epoxidase gene will be amplified using three sets of primers. The sequences will then be analyzed for point mutations using bioinformatics tools. Primers for CYP51 gene which codes for 14α-demethylase will be designed. Cycling conditions will be optimized.This target gene will be amplified and sequenced.
The sequences will then be analyzed for mutations using bioinformatics tools.
Study Design
- Study Type
- Observational
Eligibility Criteria
- Ages
- 18.00 Year(s) to 99.00 Year(s) (—)
- Sex
- All
Inclusion Criteria
- •1.All the patients above 18 years of age with suspected dermatophytic skin infection willing to give consent will be included in the study.
Exclusion Criteria
- •below 18 years those unwilling to give consent.
Outcomes
Primary Outcomes
Identification of species of dermatopytes and drug resistance in dermatophytic skin infection by rapid methods1
Time Frame: 2 weeks
Secondary Outcomes
No secondary outcomes reported