DNA fingerprinting using Diversilab system for genotyping characterization of Microsporum audouinii and Trichophyton violaceum

Objectives: To investigate the epidemiological determinants responsible for the high number of anthropophilic dermatophytes received by the National Reference Center for Mycosis of Liege (NRCL) during the year 2012. To perform a genotypic characterization by the Diversilab® system focusing on the two main isolated species, Microsporum audouinii and Trichophyton violaceum. To present a preliminary study preceding the national survey launched in 2013.
Methods: A total of 51 strains of M. audouinii (50 clinical + 1 reference (ref.) strains) and 15 strains of T. violaceum (14 clinical + 1 ref. strain) originating from different locations through Belgium were included in the study. The fungal strains were first cultivated on Malt agar, then sub-cultured in Sabouraud liquid medium (Fluka). The grown mycelium was processed for DNA extraction following recommendations of the manufacturer (Ultra Clean® DNA Microbial isolation kit, MoBio laboratories). Genotypic analysis was performed using the DiversiLab® system (BioMérieux) for DNA fingerprinting and analysis.
Results: Regarding M. audouinii, four different genotypic groups of strains were separated. Group 1 includes 11 strains and is only found in the Liège surroundings. Group 2 includes only one strain with little differences compared to group 1 and collected from the Liège area. These two groups may be related to each other. Group 3 contains 36 strains and the reference strain. This genotype is distributed in different Belgium locations. The last group, group 4, contains only 3 isolates sharing low similarities in comparison with the 3 other groups. Concerning T. violaceum, 6 different genotypic groups with a mixed geographical distribution were determined. Group 1 includes 8 clinical isolates and the ref. strain. The other five isolates are all different and seem not to be related to each other.
Conclusion: The automated typing DiversiLab® system proved to be an easy and efficient method to investigate the molecular epidemiology of dermatophytes infections. Preliminary results of the study show that, through Belgium, several groups of isolates co-exist for M. audouinii and T. violaceum providing evidence of genetic heterogeneity. This variation can be related to acquired mutations due to environmental adaptation. Further investigations are necessary to better understand the impact of this genotypic variation.