Journal de Mycologie Médicale (2015) 25, e1—e9

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

Terbinafine susceptibility and genotypic heterogeneity in clinical isolates of Trichophyton mentagrophytes by random amplified polymorphic DNA (RAPD) ´Etude de la sensibilite ´ `a la terbinafine par RAPD d’isolats cliniques de Trichophyton mentagrophytes M. Alipour a,*, N.A. Mozafari b a b

Department of microbiology, Karaj branch, Islamic Azad university, Karaj, Iran Department of microbiology, Iran university of medical sciences and health services, Tehran, Iran

Received 9 April 2014; accepted 16 September 2014 Available online 30 January 2015

KEYWORDS Random amplified polymorphic DNA technique; Dermatophytosis; Trichophyton mentagrophytes; Terbinafine

Summary Objective of the study. — The four RAPD systems tested in the present study have aimed at investigating DNA fingerprinting of Trichophyton mentagrophytes strains and the correlation between genotyping and antifungal susceptibility to terbinafine. Patients. — Twenty-nine clinical isolates of T. mentagrophytes were recovered from patients suspected of having active dermatophytosis who were referred to the laboratory of medical mycology department in Tehran university. Then, they were subjected to conventional examination by performing direct microscopic examination, culture on primary media, physiological tests. Materials and methods. — The in vitro antifungal susceptibility of twenty-nine T. mentagrophytes isolates against terbinafine was evaluated by modified agar dilution method to determine the minimum inhibitory concentration (MIC). Twenty-one sensitive and eight resistant to terbinafine, were submitted to RAPD using 4 decamer primers (A, B, C, D) with the purpose of encountering a genetic marker to terbinafine sensibility and resistance. The UPGMA-Jaccard’s correlation coefficient was used to build up dendogram that could represent clusters of similarity. According to their correlation coefficient, the samples were classified as much related (100%), moderately related (80%) and unrelated (< 70%).

* Corresponding author. E-mail address: [email protected] (M. Alipour). http://dx.doi.org/10.1016/j.mycmed.2014.09.001 1156-5233/# 2015 Published by Elsevier Masson SAS.

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M. Alipour, N.A. Mozafari Results. — All amplifications revealed distinct polymorphic bands and a total of 34 band positions was scored (0/1) for the 4 primers tested. Genetic distances between each of the isolates were calculated and cluster analysis was used to generate a dendrogram showing relationships between them. The combined dendrogram at an average similarity value of 65% grouped all strains into 2 (A, B) groups corresponding to their susceptibility reactions to terbinafine. All susceptible samples were properly grouped, but a few numbers of resistant isolates were also included. Nevertheless, further biochemical and molecular biological studies will be required to fully elucidate the point that resistance might be the result of a mutation in the gene encoding squalene epoxidase in T. mentagrophytes. Conclusion. — This study proved efficacy of applying RAPD molecular technique to complement traditional mycological culture and drug susceptibility tests for accurate and appropriate management of recurrent dermatophytosis and highlights the need for newer antifungals that can combat the emergence of terbinafine-resistant T. mentagrophytes strains. # 2015 Published by Elsevier Masson SAS.

MOTS CLÉS ADN polymorphe amplifié (RAPD) ; Dermatophytosis ; Trichophyton mentagrophytes ; Terbinafine

Re ´sume ´ L’objectif. — La présente étude est consacrée à rechercher une relation entre la résistance à la terbinafine et la parenté génétique des isolats cliniques de Trichophyton mentagrophytes en appliquant une RAPD-PCR. Les malades. — Dans cette étude, nous avons utilisé 29 isolats de T. mentagrophytes obtenus à partir des patients atteints de dermatophytose et conservés au laboratoire de mycologie médicale du département des sciences médicales à l’université de Téhéran. Les identifications ont été affirmées par les examens microscopiques, macroscopiques et les tests biochimies et physiologiques. Mate´riels et me´thodes. — La sensibilité aux médicaments des 39 exemples (isolats) cliniques T. mentagrophytes a été vérifiée en appliquant la méthode de dilution en gélose modifiée et on a désigné la concentration minimale inhibitrice (MIC). Ensuite, en appliquant quatre indices (A, B, C, D), nous avons vérifié la relation éventuelle entre le patron de résistance et de la sensibilité terbinafine avec leur parenté génétique dans 21 épreuves sensibles et 8 épreuves résidentes à la terbinafine. Selon le résultat de la matrice de similitude obtenu par la corrélation UPGMAJaccard et l’analyse spiciforme et à l’aide de UPGMA, nous avons tracé le dendrogramme des indices. Re ´sultats. — Tous les indices appliqués ont montré les différents échantillons de polymorphiques. Au total, 34 bandes d’ADN ont été faites pour 29 isolats de T. mentagrophytes. Selon le dendrogramme obtenu avec une valeur moyenne de similarité de 65 %, les isolats sont classifiés en deux catégories (A, B) d’après le patron de sensibilité au Trichophyton. Tous les échantillons sensibles ont été classés correctement, à l’exception de quelques souches résistantes. Conclusion. — La comparaison entre le patron de résistance ou de sensibilité à la terbinafine montre l’existence d’une relation génétique possible pour certains isolats en utilisant la technique RAPD. Cependant, prouver cette allégation a besoin de recherches plus complètes en études moléculaires et biochimiques. # 2015 Publié par Elsevier Masson SAS.

Introduction Dermatophytes taxonomically are divided into three genera (Epidermophyton, Microsporum and Trichophyton) that colonies keratinised tissues (skin, hair and nails) of man and animals. The colonization process is facilitated by the release of various proteolytic and other enzymes by dermatophytes, which in turn provoke inflammatory responses in the host, resulting in dermatophytosis. Trichophyton is particularly important and represents the most frequently isolated of all dermatophytes from human patients. Although infections by Trichophyton are usually restricted to the superficial epidermis, these fungi may be invasive and cause a severe and disseminated infection in immune compromised patients, such as those

associated with AIDS, diabetes mellitus, organ transplantation with the development of dermatophytic granulomas [33,41,44]. Thus, the identification of genetic features in Trichophyton strains resistant to antifungal agents might help considerably in the treatment and prophylaxis of dermatophytosis. Terbinafine is a generic antifungal agent used to treat superficial mycoses such as dermatophyte [1,39]. Terbinafine interferes with ergostrol biosynthesis by inhibiting a membrane-bound squalene epoxidase [35]. Inhibition of squalene epoxidase results in ergostrol deficiency with the accumulation of squalene, which may be responsible for in vitro fungicidal activity [15,35]. Although terbinane is widely used to treat infections caused by dermatophytes and other fungal pathogens, some of these infections are

Terbinafine susceptibility and genotypic heterogeneity in clinical isolates still difficult to resolve completely and remissions and relapses are often observed [7,17]. So far, remissions and relapses have been attributed to the inability of the antifungal drug to penetrate the site of infection rather than to the intrinsic or acquired resistance of the fungus. On one hand, the emergence of allylamine-resistant dermatophyte strains as the result of use and occasional overuse of terbinafine had gradually become prominent especially in the case of patients with onychomycosis (receiving prolonged therapy and high concentration of drug) [32]. On the other hand, routine antifungal susceptibility testing is not usually performed in the case of dermatophyte infection in clinical laboratories. So it is likely that resistance occurs but it is not detected. Morphology-based identifications of hyphae directly from the lesion samples have been used to diagnosis of dermatophytes for many years. Although they are economic, these procedures suffer from drawbacks of being non-specific so that, up to 15% of false-negative results can be attributed to this technique [36]. Also, in vitro culture is time-consuming and for some unusual and atypical isolates, identification can be very slow and may take weeks to produce a definite result [26]. Also, many dermatophytes share common genetic structures and show similar cultural characteristics [16,24]. Despite their close genetic relationship, various dermatophyte fungi have enough differences at the molecular level to be exploited for the rapid identification of several common dermatophyte species [25]. A large number of molecular techniques have been developed for identification of dermatophytes to the species level. This is due to speed, simplicity degree of handling and reproducibility of these techniques as compared to the traditional techniques. Several molecular typing methods, such as restriction fragment length polymorphism analysis of mitochondrial DNA [6,21], sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA [14], sequencing of protein-encoding genes [20,19], and arbitrarily primed PCR [AP-PCR] [27,26], and PCR fingerprinting [13], have led to dramatic progress in distinguishing closely related species and strains of dermatophytes with different profiles of virulence factors, susceptibility to antifungal drugs as well as determining whether the original isolate is responsible for reinfection or a new strain has been acquired. However, most of these techniques (e.g., restriction fragment length polymorphism analysis) are complex, laborious, time-consuming, and not easily employable for routine identification of dermatophytes; RAPD technology is simple, rapid, and, in the absence of specific nucleotide sequence information for the many dermatophyte species, able to generate species-specific or strain-specific DNA polymorphisms on the basis of characteristic band patterns detected by agarose gel electrophoresis. Randomly amplified polymorphic DNA (RAPD) methods have frequently been used for phylogenetic analysis and identification of dermatophytes. Gräser et al. [13] used four non-specific primers, (AC) 10, (GTG)5, M13 core sequence, and AP3 for 26 species of dermatophytes, of which 17 species showed characteristic patterns. They suggested that these patterns could be useful for the identification of clinical isolates even if they did not have typical phenotypic characteristics. In case of Trichophyton, Kim et al. succeeded in distinguishing T. rubrum, T. mentagrophytes by RAPD [22]. For identification of T. mentagrophytes and T. tonsurans,

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Kac et al. [18] proposed a RAPD method, in which T. mentagrophytes was divided into subtypes on the basis of amplification patterns in RAPD using three primers. These results suggest that the RAPD method is useful for identification and subtyping of dermatophytes. In this work, at first, the in vitro antifungal susceptibilities of twenty-nine T. mentagrophytes isolates against terbinafine was determined by agar dilution method as described by Mota et al. [38] with slight modifications. This important methodology used for in vitro testing of dermatophytes is simple, inexpensive, and does not require specialized equipment. Though, no agar-based susceptibility testing method has been standardized for the testing of dermatophytes. Afterward, 21 sensitive and 8 resistant to terbinafine was subjected to random amplified polymorphic DNA (RAPD) analyses, applying four single short primers, with the purpose of determining their genetic relatedness and terbinafine susceptibility and resistant of T. mentagrophytes isolates. As Trichophyton species have innate pathogenicity as well as an almost universal sensitivity to terbinafine in the past, identification to the species level associated to the evaluation of terbinafine sensitivity or resistance might be relevant for clinical management.

Material and methods Clinical materials Twenty-nine clinical T. mentagrophytes isolates were obtained from lesions of patients having active dermatophytosis, who were referred to the medical mycology laboratory department in Tehran University of medical sciences, Iran.

Preparation of fungal colonies The samples were grown on Sabouraud’s dextrose agar (Merck, Germany) supplemented with chloramphenicol and cyclohexamide 0.05% (w/v), and incubated at 28 8C for 10 days. These clinical isolates were identified to the species level by using routine phenotypic methods, including colony morphology, microscopy, physiologic, and biochemical tests [44].

Antifungal susceptibility test Terbinafine hydrochloride was obtained from its respective manufacture (Tehran chemistry), in pro-analysis pure powder form. In order to obtain the desired dilution, 1 mg of terbinafine was dissolved in acetone 50% (50 cc Acetone + 50 cc deionized water) as a stock solution of 1000 mg/ mL. The working solution was prepared by 1 in 1000 acetone 50% dilution. Under aseptic conditions, serial dilutions of the terbinafine were mixed with approximately 20 mL of warm, autoclaved liquid agar in long sterile test tubes located in bath water at 50 8C to obtain the final concentration of: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 mg/mL. They were subsequently transferred to sterile petri plates (150  6 mm) and allowed to cool and harden before using. Hardened plates were stored at 4 8C for up to 1 week prior to use. Each series of plates (containing various amount of drug) had two drug-free growth controls, one with the media alone

e4 (growth control) and the other with media including an equivalent amount of solvent used to dissolve the antifungal drug (solvent control) (Fig. 1).

Test method Using a sterile puncher, 6-mm diameter circles cut from the fresh fungal colony plaques and later transferred to the culture media in equidistant points under aseptic conditions. The plates incubated upside down at 30 8C for two weeks. The level of sensitivity to terbinafine of each fungus sample was measured by the mean diameter (mm) of the growth zone formed around colony plaques after 7 and 14 days of inoculation respectively. Each isolate was classified into: sensitive, or resistant to the drug. In order to evaluate the reproducibility of our method, new inoculum was prepared for each replicate, all isolates were run in duplicate and the standard deviations were determined. The determination of the isolates as susceptible or resistant is complex and not yet been established for dermatophytes, but high MIC values were found for some isolates. The degree of inhibition growth was made visually by comparing the mean growth (mm) in the plates containing the drug with the growth in the drug-free 3 control plates and reported as inhibition growth according to the following formula: The highest dilution of the drug, which thoroughly inhibited fungal growth, was taken as the MIC. MICs determined were stable at least up to 96 h. MIC50 was calculated by taking the drug concentration, where 50% of isolates are inhibited. Similarly, MIC90 was noted with drug concentration where 90% of the isolates were inhibited compared to the growth of the corresponding growth control. The significance of differences in mean values was determined using Student’s t test. P values < 0.05 0.05 were considered statistically significant.

Figure 1 The efficacy of terbinafine against 29 clinical strains of Trichophyton mentagrophytes on subouraud’s dextrose agar using modified agar dilution method. S: media without drug (growth control); C: medium with acetone 50% and no drug (soluble control); A to I containing 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 mg/mL of terbinafine respectively. ´ de la terbinafine sur 29 isolats cliniques de TrichoEfficacite phyton mentagrophytes sur milieux de Sabouraud dextrose agar ´ thode de dilution en en utilisant une modification de la me ´ lose. S : milieu sans antifongique (te ´ moin croissance) ; C : ge ´ tone 50 % et sans antifongique (te ´ moin solvant) ; milieu avec ace A `a I : milieu avec 0,0005, 0,001, 0,005, 0,01, 0,05, 0,1, 0,5, 1 et 5 mg/mL de terbinafine.

M. Alipour, N.A. Mozafari

DNA extraction Genomic DNA was extracted as described by Del Sal et al. [8] with a few modifications. In brief, 1—2 g of a 7-day-old mycelium, grown on SDA-CC agar was harvested by sterile scalpel and washed several times by 0.1 M MgCl2. Liquid nitrogen was added to 2 to 3 g of frozen hyphae in a prechilled mortar and the cells were ground finely with a pestle. Approximately 200 mg of frozen, ground mycelium was placed in a 1.5 mL micro-centrifuge tube containing 500 mL of lysis buffer (50 mM Tris, pH 8.0, 50 mM EDTA, pH 8.0, 250 mM NaCl, 0.3% [wt/vol] sodium dodecyl sulfate [SDS], pH 8.0), and 500 mg of acid-washed 0.4—0.6 mm diameter glass beads. Samples were incubated for 1 h at 60 8C with occasional mixing, then 100 mL of 5 M sodium perchlorate was added and incubation continued for a further 15 min at 60 8C. The solution was treated with RNase A (Cinnageninc, Iran) at a final concentration of 50 mg/mL for 1 h at 37 8C. Tubes were cooled on ice, and extraction was performed with 500 mL of ice-cold chloroform followed by adding equal volumes of phenol-chloroform-isoamyl alcohol (25:24:1; pH 8.0) and finally with chloroform. Purified nucleic acids were precipitated with 2 volumes of ice-cold 75% ethanol, washed twice in 500 mL of 70% ethanol, air dried, and resuspended in 40 mL of sterile water.

RAPD assay The following decamer oligonucleotides of arbitrary sequence were used as single primers in the RAPD experiments are:    

primer primer primer primer

A50 -d [GTGACGTAGG]-30 ; B50 -d [ATGGATCCGG]-30 ; C 50 -d [CAGAAGCCCA]-30 ; D 50 -d [(GACA)4]-30 .

Amplification reactions were performed in final volumes of 50 mL containing 25 ng template DNA, reaction buffer (10 mM Tris-HCl [pH8.3], 50 mM KCl), 2.5 mM MgCl2, 200 mM (each) dATP, dCTP, dGTP, and dTTP, 160 ng of each primer (Cinnageninc., Iran) and 2.5 U of Taq DNA polymerase (Fig. 2). The samples were overlaid with sterile paraffin oil (Sigma) and PCR was performed for 35 cycles in a DNA thermal cycler (Armin tebco, Iran). An initial denaturation of 4 min at 94.5 8C was followed by 35 cycles consisting of denaturation for 30 s at 94 8C, annealing for 1 min at 30 8C and extension for 1 min at 72 8C, and then a final extension for 1 min at 72 8C. A total of 10 mL of each amplification product was separated by electrophoresis on 1.8% agarosegels TAE (4.84 g Tris base, 1.14 g glacial acetic acid, 2 mL 0.5 Na EDTA PH.8) gels for 2 hours and subsequently visualized by Nickon coolpix 4500 digital photograph machine after ethidium bromide (0.5 mg/mL) staining. Marker (Gene ruler 100 bp, QiaGene, Germany) was included for size estimation of the profiles. Only intense and reproducible fragments were considered for analysis amplification on two different occasions. All amplifications were done with rigorously standardized concentration of reagents, the same thermal cycler and the same cycling conditions. RAPD profiles showed identical band patterns and reproducibility was 100% (data not shown).

Terbinafine susceptibility and genotypic heterogeneity in clinical isolates

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Figure 2 A—D. Electrophoretic separation of RAPD-PCR products amplified form twenty-nine T. mentagrophytes isolates obtained with four different primers. Lanes M, 100 bp ladder marker (QiaGene) 1—29, RAPD-PCR products generated from 1— 29 T. mentagrophytes isolates. ´ paration ´electrophore ´ tique des produits amplifie ´ s par RAPD-PCR de 29 isolats de T. mentagrophytes obtenus avec 4 primers A—D. Se ´ ne ´ re ´ s par RAPD-PCR de 1—29 isolats de ´ rents. Bandes M, ´echelle marqueur pour 100 pb (QiaGene) ; 1—29 : produits ge diffe Trichophyton mentagrophytes.

The selection of primers The selection of appropriate primer and optimization of PCR conditions are of great importance for maximizing the discriminatory power and reproducibility of RAPD analysis. Moreover, the standardization of DNA template concentration is of significant importance for avoiding artifact band patterns [8]. Primers A, B were chosen from the primers used in RAPD technique in differentiating of T. mentagrophytes by Yazdanparast et al. [45]. Primer C was selected from the primers used in RAPD research by Shahnavaz et al. [42] and primer D was selected from the primers used in RAPD research by Atef et al. [2].

Analysis of RAPD patterns In the present investigation, we initially chose over eleven random decamer primers to amplify DNA polymorphism profile, each of these primers multiplied a different profile and produced distinct bands. We chose four of these primers for further study as they produced consistent and reproducible bands for all of the fungal isolates. All visible and welldefined bands were identified visually and confirmed by software.

Phylogenic analysis For considering a marker as polymorphism, the absence of an amplified product in at least one sample was used as a criterion. The RAPD-PCR used in this study yielded myriad bands in the agarose gel upon electrophoresis. The number and frequency of unique bands produced a specific DNA sample compared to all the other samples, were determined.

A 100 bp ladder was run tougher with PCR products as a molecular weight marker. PCR banding sizes generated with each primer were measured according to relative motion of bands by SEQUID software with accuracy of 1/20 millimeter. Positions of bands were conveyed into a binary character matrix ‘‘1’’ and ‘‘0’’ for the absence of band was recorded at a particular position. The NTYSYS-PC software 2.02 was used to estimate genetic similarities with Jaccard’s coefficient. The matrix of generated similarities was analysed by unweighted pair group method with arithmetic average (UPGMA), using the SAHN clustering module. The cophenetic correlation coefficient was applied to compute a cophenetic value matrix using the UPGMA matrix. The MXCOMP module was then used to calculate the cophenetic correlation, to test the goodness of fit of cluster analysis to the similarity matrix. The cophenetic correlation coefficient obtained in this survey was 0.98 showing very goodness of fit of the cluster analysis to the similarity [3] (Fig. 3).

Results Culture results The in vitro susceptibility of 29 T. mentagrophytes isolates to terbinafine is summarized in Table 1. The MIC50 and MIC90 are also shown. The data are presented as MIC ranges and, where appropriate, as the drug concentrations required to inhibit 50 and 90% of the isolate of each species (MIC50 and MIC90 respectively). The results of experimental indicate that the concentration of terbinafine required for complete growth inhibition of T. mentagrophytes isolates was observed in the range of 0.1 to 5 mg/mL and no growth was observed in any of the isolates over the concentration of

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M. Alipour, N.A. Mozafari

Figure 3 The cophenetic correlation coefficient was applied to compute a cophenetic value matrix using the UPGMA matrix. ´ fficient de corre ´ lation cophe ´ ne ´ tique a ´ete ´ utilise ´ pour Le coe ´ ne ´ tique en utilisant la calculer une valeur de matrice cophe matrice UPGMA.

Table 2 shows the total number of amplified fragments and the number of polymorphic fragments produced with each primer. A dendrogram based on the combined similarity matrix generated with the four RAPD primers is presented in Fig. 4. The combined dendrogram at an average similarity value of 65% grouped all strains into 2 (A, B) clusters showing high diversity in profiles. The first group, Group A included 21 isolates (1, 2 . . . 7, 8) had 69.2% similarity and were distributed into two subgroups, IA and IIA, which included 23 (79.3%), of the isolates. All isolates were sensitive to terbinafine (except 27, 29 isolates which were resistant to terbinafine). The ten isolates strongly related genetically had > 92% similarity. Five of these had similarity coefficients of 100%, being genotypically identical with an SJ value of 100. Group B included 8 isolates (9, 10 . . . 27, 28) and were distributed into two subgroups, IB and IIB, at similarity value of 80.8%. This group included 6 (20.7%) of the isolates. The five isolates strongly related genetically had > 85% similarity. Three of these had similarity coefficients of 100%, being genotypically identical with an SJ value of 100.

Reproducibility and stability of the methods 5  mg/mL (P < 0.05). The growths of 21 isolates (62%) were completely inhibited in 0.1—1 mg/mL of terbinafine while 8 isolates were remaining resistant. Thus susceptibility test data confirmed that these eight isolates had a primary trend of increasing terbinafine MICs resulted in increased resistance.

Molecular typing The RAPD with four primers generated 13 monomorphic bands and 21 polymorphic bands in a total of 34 banded ranging from approximately 300 to 1800 bp in length. RAPD patterns for the T. mentagrophytes isolates applying primer A, C were very distinct from the profiles using primer B, D.

To evaluate the stability of the methods, each sample of genomic DNA was amplified in duplicate in repeated PCRs at different times. To test for reproducibility, all amplifications were done with rigorously standardized concentrations of reagents, the same thermal cycler and the same cycling conditions. RAPD profiles showed identical band patterns (data not shown) and reproducibility was 100%.

Discussion Dermatophytoses are the most frequent fungal infections worldwide, affecting individuals in various age groups and leading to these patients’ low quality of life and economic

Table 1 Susceptibility data for 29 isolates of Trichophyton mentagrophytesas against terbinafine. ´ de 29 isolats de Trichophyton mentagrophytes `a la terbinafine. ´ sultat de la sensibilite Re Antifungal agent

Terbinafine

Type of T. mentagrophytes isolates (no. of isolates tested)

Terbinafine-resistant (n = 8) Terbinafine-sensitive (n = 21)

GD (mm)

MICrange

mg mL




MeanTER (GD)  SD (mm)

GD range (mm)

MIC50

MIC90

Range

64.33  7.01 28.65  7.53

47—70 15—45

0.5 0.05

5 0.1

0.5—5 0.05—0.1

GD: growth diameters (GD); MIC: minimal inhibatory concentration; MIC50 and MIC90: MIC inhibiting 50% and 90% of the isolates.

Table 2 Characteristic of primers used in this survey. ´ s dans cette ´etude. ´ ristiques des primers utilise Caracte Primers

Nucleotide sequences

Produced bands

Polymorphism bands

Length of produced bands (bp)

A B C D

5’-GTGACGTAGG-3’ 5’-ATGGATCCGG-3’ 5’-CAGAAGCCCA-3’ 5’-(GACA)4-3’

11 5 10 8

8 2 6 5

400—1800 565—1080 560—1310 300—650

Terbinafine susceptibility and genotypic heterogeneity in clinical isolates

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Figure 4 Dendrogram of RAPD system from the 29 T. mentagrophytes isolates. The UPGMA-Jaccard’s correlation coefficient was used to build up dendogram. The dendogram representing the cluster of similarity were classified according to the degree of genetic relatedness. 1.0 or 100% for identical samples; 0.9 or 90% for very related samples; 0.8 or 80% for moderately related samples;  70% for non-related samples (R: isolates resistant to terbinafine, S: isolates susceptible to terbinafine). ´ lation UPGMA-Jaccard a ´ete ´ utilise ´ pour ´etablir le Dendrogramme de RAPD de 29 isolats de T. mentagrophytes. Le coefficient de corre ´ sentant les groupes de similarite ´ ont ´ete ´ classe ´ s selon le degre ´ de relation ge ´ ne ´ tique : 1 ou dendrogramme. Les dendrogrammes repre ` s proches ; 0,8 ou 80 % pour des ´echantillons mode ´ re ´ ment 100 % pour les ´echantillons identiques ; 0,9 ou 90 % pour des ´echantillons tre ´ s ;  70 % pour des ´echantillons non lie ´ s (R : isolats re ´ sistants `a la terbinafine, S : isolats sensibles `a la terbinafine). relie

burden due to treatment expenditures. Research about different aspects of dermatophytes, such as physiology, genetics, and biochemistry, as well as the pathogenesis of dermatophytoses and the immune response triggered by these infections, are essential to the development of new therapeutic and prophylactic measures. In this study, we tested the antifungal susceptibility of 29 isolates of T. mentagrophytes. Although a broth microdilution adaptation of the NCCLS reference, method for yeast has shown good intra- and inter-laboratory reproducibility [11,10]. There is no standardized method for susceptibility testing of filamentous fungi. In addition, these methods are expensive and need specific media and equipment such as RPMI, MOPS buffer, and micro-plate trays [34]. For this reason, we used agar-based dilution method that enables determining the efficacy of various antifungal drugs against various fungal genera and species. We found that the MIC ranges for dermatophytes against terbinafine was similar to those previously found by several researchers [39,33,5]. Therefore, this assay can be adapted for routine diagnosis in the laboratory and for assessment of dermatophyte resistance against antifungal drugs. As genotypic analysis of the isolates before and after treatment of patients was not performed, so it was not possible to determine whether the high MICs of T. mentagrophytes isolates found in the present study correlate to innate or acquired traits of the isolates, although innate resistance seems less likely given the variable frequency of high MICs. Another probable explanation for acquired resistance could be selection pressure, which might occur during long drug therapy. Namely, is a pool of some closely related strains that coexist and at any proper time one strain take advantage and undergoes progressive minor genetic variations and becomes dominant. These microevolutionary changes within an infecting strain over time have been demonstrated in Candida albicans [43].

So it is possible, due to micro-evolution, that some substrains in colonizing population present altered phenotypes, including changes in susceptibility to antifungal drugs. One can suggest, in a patient with chronic dermatophytosis, that high population density of T. mentagrophytes is a critical factor for strain microevolution or colonal selection of resistant substrains under the drug selection pressure. This situation is probably more significant in patients with onychomycosis who have a long-term therapy with antifungal drugs. Generally, the main biochemical and molecular mechanisms that contribute to the drug resistance phenotype in fungi are: reduction of drug delivery, metabolic modification or degradation of the drug by the cell, alterations in the interaction between the drug and the target site or other enzymes involved in the same enzymatic pathway, through punctual mutations, overexpression of the target molecule, gene amplification and conversion (recombination); increase of the cell efflux; for instance, through greater expression of efflux pumps such as ATP binding cassette transporters. The resistance of dermatophytes to inhibiting agents involves the participation of targetenzyme modifiers, overexpression of ATP binding cassette transporters and stress-related proteins [41]. Previous studies have demonstrated terbinafine resistance of dermatophytes variants directly related to application of the drug. In 1999, a modified squalene epoxidase with reduced affinity for terbinafine conferred terbinafine resistance to Nectria haematococca mutants [23] and, in another, a Candida albicans strain was resistant to terbinafine due to activation of the multidrug efflux transporter CDR2 [40]. Also, a, C. albicans mutant carrying CDR1 deletion resulted in terbinafine hyper-susceptibility [40]. In Aspergillus nidulans overexpression of salA, gene encoding salicilate1-monoxygenase enzyme was possibly responsible for degrading of terbinafine [30]. In T. rubrum, two ATP binding cassette transporters, TruMDR1 e TruMDR2, were identified showing importance not only in the process

e8 of resistance to various antifungal drugs, but also in enzyme secretion and probably in the pathogenicity of this dermatophyte [4,12,29]. It has also been described that a mutation in the gene that codifies the enzyme squalene epoxidase (ErgA), target of terbinafine, made the fungi Aspergillus nidulans, A. fumigatus and T. rubrum highly resistant to this drug [31,37]. However, in 2003, a case of clinical resistance to terbinafine, one of the most used drugs to treat dermatophytoses caused by T. rubrum, was reported. This resistant lineage was isolated from a patient with onychomycosis whose oral treatment with terbinafine was ineffective, showing cross-resistance to various other inhibitors of squalene epoxidase, including naftifine, butenafine, tolnaftato and tolciclate, suggesting a target-specific resistance mechanism [32]. But little is known about the mechanisms of resistance in T. mentagrophytes. So the elucidation of genetic traits by which fungi can exert resistance to terbinafine is of great interest. If resistant isolates do emerge, typing methods provide new insights into pathogenicity of these fungi. Also, comprehension of the events that promote resistance is essential for the development of structural modifications in the antifungal drugs currently used in medical practice. The four RAPD systems tested in the present study have aimed at differentiating T. mentagrophytes according to their genetic distance and also with respect to terbinafine sensitivity or resistance. RAPD analysis has demonstrated enormous success in fungal infection studies [9]. On one hand, the main problem is the reproducibility, not only among laboratories, but also within a laboratory over time. Artifactual variation can occur as a result of small differences in the primer: template concentration ratio, the temperatures during amplification and the concentration of magnesium in the reaction mixture [28]. To contrast to most of the results in the literature, in this study, we showed identical band patterns and a reproducibility of 100% (data not shown). These results were obtained from a fixed mixture, with standardized concentrations of the reagents and the same thermal cycler for all the RAPD reactions. On the other hand, RAPD cannot be used to determine dermatophytes species directly from biological samples without prior culture isolation and above all, the DNA fragments generated by RAPD, although covering the entire fungus genome, are not associated to any of the seven proposed (previously mentioned) mechanisms leading to allyalmines compounds resistance. In our study, the RAPD systems with four primers might possibly be used for this application, as they have clustered the resistant isolates in a few closely related dendograms. We concluded that primers B, D can partially discriminate between sensitive and resistant to terbinafine isolates, but might eventually be used to determine species. The primers A and C have been shown to provide a high level of determination between strains of T. mentagrophytes with respect to sensitivity to terbinafine. Although the exact genetic structures and functions of the characteristic bands amplified from Trichophyton mentagrophytes with the random primers are not clear at the moment, however, it is anticipated that the ongoing analysis of the nucleotide base sequences of these bands will provide further insights

M. Alipour, N.A. Mozafari into the genetic structures and possible functions of the gene regions involved, leading to the development of new strategies of diagnosis, therapy and prevention of dermatophytoses.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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