Epidemiological and Molecular Analysis of Scalp Ringworm in France (TCC-France)

Objectives/Hypotheses The overall objective of this study is to describe the epidemiology of tinea capitis (TC) in France from clinical, species, and treatment response perspectives.

To achieve these objectives, it will be necessary to:

• Identify isolates using various methods (morphological, proteomic, and molecular),
• Perform phylogenetic analysis of the isolates,
• Determine the susceptibility of isolates to four antifungal agents (terbinafine, itraconazole, voriconazole, and ketoconazole),
• And, in the event that resistant isolates are identified, characterize the mechanisms of resistance to terbinafine and/or azoles.

Methodology Participating Centers Eleven centers (10 university hospitals and one private laboratory) were selected based on the results of two national surveys on TC conducted by the French Society for Medical Mycology (SFMM) between 2014 and 2019. Centers reporting at least 20 cases per year were included, ensuring at least one center per region. The estimated number of cases for 2023 is approximately 635.

The participating centers will be responsible for:

• Identifying dermatophyte isolates from scalp samples using classical phenotypic methods and/or MALDI-TOF mass spectrometry with the online MSI-2 database (for the 6/11 centers that routinely use this method),
• Storing isolates in microtubes at -20°C (or sending primary culture tubes monthly for the five Parisian centers) for subsequent shipment to the Parasitology-Mycology Laboratory in Bobigny,
• Completing part of the data collection form (using a request sheet filled out at the time of sampling, provided as an appendix).

Isolates All dermatophyte isolates obtained from scalp sample cultures in the 11 centers between January 2, 2023, and December 31, 2023, will be studied. After identification using each laboratory's standard techniques, isolates will be stored at -20°C in microtubes (provided) for later shipment to the Parasitology-Mycology Laboratory at Avicenne Hospital. Parisian centers may choose to send primary culture tubes monthly or to store isolates in microtubes at -20°C for later shipment to Avicenne.

Data Collection Form A REDcap collection form will be available online. Isolates will be anonymized using a code for each center, consisting of a three-letter abbreviation for the city or hospital followed by a number (001 to XXX).

Each center will record the following data: local case number, demographic and clinical information, phenotypic identification (with or without MALDI-TOF MS), previous treatments (corticosteroids and/or antifungals), first-line treatment (dose and outcome at one and, if necessary, two months-cure/improvement or need for second-line treatment).

Additional results (MALDI-TOF MS identification, ITS region sequencing, and antifungal susceptibility testing) will be entered by the coordinating center.

Patients will be required to sign a non-opposition form for the use of their demographic, clinical, and biological data for research purposes. All procedures will adhere to the ethical standards of the 1975 Declaration of Helsinki, as revised in 2008.

Isolate Identification Dermatophyte isolates will be identified phenotypically by each center and by MALDI-TOF MS using the online MSI-2 database (https://msi.happy-dev.fr) for centers that already use this method routinely, or by the coordinating center. Molecular identification will also be performed by the coordinating center using PCR-sequencing of ribosomal DNA targeting the ITS1, 5.8S rDNA, and ITS2 regions, as well as the β-tubulin 2 gene (14), following fungal DNA extraction using the Chelex method. The obtained sequences will be aligned with reference sequences from various databases (MycoBank, ISHAM Barcoding, and GenBank).

Phylogenetic Analysis of Isolates A phylogenetic analysis will be performed for the predominant species isolated, presumably T. tonsurans, representing approximately 30-35% of isolates. This analysis will use maximum likelihood methods via MEGA software, based on sequences of the ITS regions and the β-tubulin 2 (BT2) gene. Additional genes such as ALP1, SQLE, and TEF-1α may also be sequenced for multi-locus sequence typing (MLST) analysis (8,15).

Other isolates will be preserved and studied as part of a doctoral thesis, but as an ancillary study to this research project.

Antifungal Susceptibility Testing For non- or poorly sporulating isolates (such as Microsporum audouinii and species within the T. rubrum complex), subculturing on potato dextrose agar supplemented with 20% CO₂ will be performed beforehand to stimulate sporulation (19).

Antifungal susceptibility will be determined for sporulating isolates using three different methods by the coordinating center:

• Screening for terbinafine-resistant isolates using a terbinafine-containing agar medium (0.2 mg/mL TCAM) (16). Each isolate will be cultured in parallel on RPMI agar plates with and without terbinafine, following an established protocol (A. Sabater-Moreno and E. Dannaoui).
• Determination of minimum inhibitory concentrations (MICs) using RPMI agar strips for itraconazole (E-test™ strips, BioMérieux) and terbinafine (Ezy MIC™ strips, DMLABO).
• Determination of MICs for terbinafine, itraconazole, voriconazole, and ketoconazole (Sigma-Aldrich) using the EUCAST method adapted for conidia-producing dermatophytes (17,18). The 50% inhibitory concentration (IC₅₀) will be determined by spectrophotometry. Isolates will be considered at risk of resistance when the IC₅₀ exceeds the Epidemiological Cut-Off Value (ECOFF).

Characterization of Resistance Mechanisms to Terbinafine and Azoles Various antifungal resistance mechanisms have been reported in dermatophytes, including point mutations or alterations in drug targets, as well as increased efflux of antifungals due to overexpression of ABC or MFS transporter genes, or more recently, overexpression of the TinCYP51B gene encoding lanosterol 14α-demethylase, the target of azoles (20-23).

Resistance to terbinafine is primarily associated with modifications in squalene epoxidase (SQLE), an enzyme involved in an early step of membrane ergosterol synthesis and the target of allylamines. If terbinafine-resistant isolates are identified during this study, the SQLE gene will be amplified and sequenced to detect point mutations (Leu393, Phe397, Phe415, and His440) by aligning obtained sequences with reference sequences from various databases (MycoBank, ISHAM Barcoding, and GenBank). If azole-resistant isolates are identified, point mutations in the ERG11 gene encoding sterol 14-α demethylase will be investigated.