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Evaluation of the Whole Genome Sequencing (WGS) and Ef-1 alpha/ITS1/ITS2 sequencing for T. indotineae/T.mentagrophytes/T.interdigiale typing and identification among Belgian strains.
Sacheli, Rosalie; El Moussaoui, Khalid; EGREK, Sabrina et al.
202311th Trends in Medical Mycology (TIMM) -Athens
Peer reviewed
 

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Keywords :
Terbinafine resistance, Trichophyton indotineae whole genome sequencing
Abstract :
[en] Objectives: Trichophyton (T.) indotineae is a newly identified dermatophyte characterized as near-epidemic form on the Indian subcontinent and closely related to T. interdigitale/mentagrophytes. The huge rate of resistance to terbinafine of this dermatophyte is problematic and leads to extended dermatophytoses. Terbinafine resistance has been associated with mutations on the squalene epoxidase (SQLE) gene. Currently, the identification of dermatophytes at the National Reference Center for Mycoses in Liège (NRCML) is based on microscopic examination coupled with sequencing of the internal transcribed spacer 2 region (ITS2). T. indotineae/mentagrophytes/interdigitale cannot be distinguished by ITS sequencing. So, there is a need for efficient molecular tools to easily discriminate these species. In our study, whole genome sequencing (WGS) and Ef-1 alpha/ITS1/ITS2 sequencing have been evaluated for this purpose. Methods: In total, 59 dermatophytes strains have been characterized. Among 53, from the NRCML collection (2018-2022), 34 were identified as T. mentagrophytes by conventional methods while 19 were identified as T. interdigitale. Six additional T. indotineae reference strains were included (BCCM/IHEM fungi collection, Brussels). WGS has been performed by Illumina sequencing (GIGA Genomics, Liège, NovaSeq S4 V1.5 300 cycles XP workflow). Assembly of the genome was done using SPAdes integrated in a custom made bioinformatic pipeline “WGS typer” (Hedera22, Liège). Similarity dendrogram was generated by WGS typer using the maximum likelihood method. Reference strains of T. indotineae/mentagrophytes/interdigitale were included on the tree. In parallel, Ef-1 alpha/ITS1/ITS2 region Sanger sequencing was evaluated as rapid identification tool in association with the generation of a dendrogram by maximum likelihood using RAxML-NG. Minimal inhibitory concentrations (MICs) of selected strains have been determined by the microdilution method EUCAST E.Def.11.0. Results: WGS characterized 25 strains as T. mentagrophytes, 22 as T. interdigitale and 12 as T. indotineae including the 6 reference strains. Three distinct clades, one for each species were visible on the dendrogram based on WGS results. Ef-1 alpha gene sequencing associated with phylogenetic tree generation allowed to obtain the same discrimination power than Illumina WGS to distinguish among the three species while ITS1 and ITS2 gene sequencing didn’t permit to efficiently distinguish between T. interdigitale and T. mentagrophytes. T. indotineae was part of a well distinct clade considering the three studied regions. Among the T. indotineae clade, the SQLE coding gene has been checked for amino-acids substitutions. Seven out of 12 strains (58%) shared the F397L substitution on SQLE, and 1/12 (8,3%) presented the L393F substitution. Four out of 12 strains (33,3%) didn’t harbor any substitutions on SQLE. The MICs for terbinafine of T. indotineae strains have been determined: 8 (67%) out of 12 strains (also harboring SQLE substitutions) were resistant to terbinafine with MIC values >2µg/ml. Conclusion: Our study demonstrates that since 2018, terbinafine-resistant strains of T. indotineae circulate in Belgium but were not correctly identified. The powerful discriminatory power of WGS for the genomic differentiation of T. indotineae/mentagrophytes/interdigitale species has been confirmed. Moreover, a rapid phylogenetic approach based on Ef-1 alpha gene sequencing showed to be as efficient as WGS to discriminate between the three species.
Disciplines :
Microbiology
Author, co-author :
Sacheli, Rosalie  ;  Centre Hospitalier Universitaire de Liège - CHU > > Service de microbiologie clinique
El Moussaoui, Khalid  ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Bactériologie, mycologie, parasitologie, virologie et microbiologie ; Centre Hospitalier Universitaire de Liège - CHU > > Service de microbiologie clinique
EGREK, Sabrina ;  Centre Hospitalier Universitaire de Liège - CHU > > Service de microbiologie clinique
DARFOUF, Rajae ;  Centre Hospitalier Universitaire de Liège - CHU > > Service de microbiologie clinique
Hayette, Marie-Pierre ;  Centre Hospitalier Universitaire de Liège - CHU > > Service de microbiologie clinique
Language :
English
Title :
Evaluation of the Whole Genome Sequencing (WGS) and Ef-1 alpha/ITS1/ITS2 sequencing for T. indotineae/T.mentagrophytes/T.interdigiale typing and identification among Belgian strains.
Publication date :
20 October 2023
Event name :
11th Trends in Medical Mycology (TIMM) -Athens
Event organizer :
ECMM
Event place :
athènes, Greece
Event date :
20-23 octobre 2023
Audience :
International
Peer reviewed :
Peer reviewed
Available on ORBi :
since 23 November 2023

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