First identification of Trichophyton rubrum var. raubitschekii in Constantine (ALGERIA)

Anissa Zohra Hafirassou 1 , Nadia Gassem 2 , Anne Lise Bienvenu 3 , Guillaume Bonnot 3 , Stéphane Picot 3 and Ilhem Mihoubi 1 . 1. Laboratory of Mycology, Biotechnology and Microbial Activities; Department of Microbiology, Faculty of Natural Sciences and Life, Frères Mentouri University, Constantine (Algeria). 2. Laboratory of ParasitologyMedical Mycology; CHU Ben BadisConstantine (Algeria). 3. Institut of ParasitologyMedical Mycology, Lyon, SMITH, ICBMS, UMR 5246 CNRS, Claude BernardLyon 1 University (France). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Onychomycosis is a nail infection caused by different fungal belonging to dermatophytes, yeasts and molds. Dermatophytes are the most incriminated. The aim of our study is to establish both conventional and molecular diagnosis for onychomycosis in Constantine. Sixteen nail samples were collected from patients with nail lesions, clinically suspected of onychomycosis. Direct microscopic observation and culture on Sabouraud medium with and without cycloheximide were performed. The identification was based on macroscopic and microscopic features, and confirmed by real-time panfungal PCR analysis. The ITS1-5.8S-ITS2 rDNA region was amplified using the ITS1 and ITS4 primers; and sequenced. The 16 samples were positive on their KOH direct examination. On Sabouraud medium, only 5/16 gave positive culture. The urease test on Christensen medium performed on the 5 isolates revealed a positive urease for one isolate and showed numerous macroconidia in microscopic observation. This isolate showed 99 % of homology with both Trichophyton raubitschekii JX827168.1 and Trichophyton rubrum FM178326.1 in molecular diagnosis. Based on phenotypical characteristics and molecular analysis, this isolate was identified as a Trichophyton rubrum var. raubitschekii. The ITS phylogenetic tree, showed 100 % of homology in the ITS region of Trichophyton rubrum, and Trichophyton raubitschekii isolated. The systematic use of the urease test for Trichophyton rubrum could contribute to increase the prevalence of Trichophyton rubrum var. raubitschekii in the world.
Trichophyton rubrum and Trichophyton mentagrophytes are responsible for nearly 90% of toenail and at least 50% of fingernail onychomycosis (Tanrıverdi and Özer, 2013;Yadav et al., 2015). T. rubrum represents over than 80% of nail dermatophytes (Gupta and Nakrieko, 2015), and is considered as a complex of species including multiple morphotypes. Recently, several of these morphotypes, have been formally identified as variants of T.rubrum, including T. raubitschekii, usually found in Africa, Southeast Asia, Australia and South America (Hiruma et al., 2012) with tinea corporis and tinea cruris. However, it is weakly associated with tinea pedis and onychomycosis.
Identification of causative agents responsible for onychomycosis is usually based on direct microscopic examination of clinical samples and culture in order to determine the fungal features. The development of molecular biology techniques is a valuable addition to the detection and identification of dermatophytes. In this context, we used conventional methods (direct examination and culture) and a real-time panfungal PCR assay for the diagnosis of onychomycosis in Constantine (Algeria) in the aim of deciphering T. rubrum complex looking for species closely related to this one.

Patients and methods
Nail samples:-A total of 16 nails samples were collected from 16 patients oriented by their dermatologists, consulting at the Parasitology-Mycology Laboratory of Constantine Hospital (Algeria) for onychomycosis suspicion. Patients who received a local and/or a systemic antifungal treatment, during the three months before the consultation, were excluded from the study. Nails were taken from the infected area, in the junction between infected and healthy nail zone (Chabasse and Pihet, 2014). In order to sequence the fungal DNA, we purified the amplicon using a PCR purification kit (Qiaquick PCR Purification) and sequenced it using ITS1 and ITS4 primers, according to Sanger method. Results were compared on 1749 a BLAST search via the National Center for Biotechnology Information (NCBI) nucleotide database (http://www.ncbi.nlm.nih.gov/BLAST).
A phylogenetic analysis of sequences representing the Internal Transcribed Spacer (ITS) region of rDNA amplified from T. rubrum isolated, and sequences of T.rubrum complex available in the GenBank database, were constructed with Neighbor joining method (Saitou and Nei, 1987) and with the substitution Kimura two parameters model and a 1000 replicates Bootstrap in Molecular Evolutionary Genetics Analysis-MEGA 6.0 (Kumar et al., 2008). Phylogenetic tree was produced using Trichophyton interdigitale (accession no KP308373.1) as an out-group.

Results:-
Patients' ages ranged between 23 and 75 years and the sex-ratio was 0,3. The 16 samples were positive on direct KOH examination and showed the presence of mycelium (Figure 2). On Sabouraud medium, only 5/16 samples led to positive culture. Patients had two clinical presentations: the distal lateral subungual onychomycosis (DLSO) and the DLSO hyperkeratosis with a thickened nail, brown yellowish color, peeling off at the distal portion of the nail (Figure 1). The cultures were flat to slightly raised, white to cream, sued-like to downy, with a yellow-brown to wine-red reverse. On PDA medium, the five colonies were fluffy disc-shaped with rounded edges and raised centers. Furthermore, they developed wine red pigment on their reverse ( Figure 3A and B).