Use of PCR-HRMA for Direct Detection and Identification of Dermatophytosis Agents from Clinical Samples


Authors: R. Dobiáš 1,2;  M. Kantorová 3;  P. Jaworská 1;  P. Hamal 2;  J. Mrázek 3
Authors‘ workplace: Oddělení bakteriologie a mykologie, Centrum klinických laboratoří, Zdravotní ústav se sídlem v Ostravě, vedoucí oddělení RNDr. Vladislav Holec 1;  Ústav mikrobiologie, Lékařská fakulta Univerzity Palackého v Olomouci, přednosta ústavu prof. MUDr. Milan Kolář, Ph. D. 2;  Oddělení molekulární biologie, Centrum klinických laboratoří, Zdravotní ústav se sídlem v Ostravě, vedoucí oddělení Mgr. Jakub Mrázek 3
Published in: Čes-slov Derm, 93, 2018, No. 6, p. 259-265
Category: Clinical and laboratory Research

Overview

The aim of the study was to test the utility of the PCR method in combination with High Resolution Melting Analysis (HRMA) for the detection and identification of dermatophytes directly from clinical skin and adnexa samples. The methodology should reduce the time between sampling and diagnosis, increase the diagnostic sensitivity, and overall improve patient care. In the study, 128 clinical samples from patients with suspected dermatomycosis were analyzed. To isolate fungal DNA from the clinical specimens, a KIT ZR Fungal/Bacterial DNA MiniPrepTM was used. Two sets of primers specific for a wide range of dermatophytes were used to detect ribosomal DNA regions. The real-time PCR High Resolution Melting Analysis (PCR-HRMA) method was used for dermatophyte species identification. The PCR detection success was 74% for both sets of primers, the PCR-HRMA method enabled dermatophyte species identification in all PCR positive cases. In contrast, only 52% of patients with dermatophytosis were culture positive. Microscopy from the sample was positive in 90% of patients with proven dermatophytosis. 90% of dermatophytes were successfully identified using microscopy, culture and PCR-HRMA. The most common dermatophyte species (Trichophyton rubrum, T. interdigitale, T. benhamiae) were reliably detected by this methodology. PCR detection of rDNA directly from clinical material using HRMA increased the number of species identificacions in the diagnosis of dermatophytosis. The combination of classical and molecular biologic examinations appears to be a suitable method for the rapid and reliable diagnosis of dermatophytosis.

Keywords:

Trichophyton benhamiae – dermatophytes – direct identification – ribosomal DNA – Trichophyton rubrum – Trichophyton interdigitale


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Dermatology & STDs Paediatric dermatology & STDs
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