Terbinafine is the drug of choice in the treatment of dermatophytosis due to T. rubrum and T. mentagrophytes as the most important species involved in chronic resistant infections. In 2005, Osborne et al. published the first report on terbinafine resistance in T. rubrum2. Increasing numbers of these reports are worrisome in medical society and are becoming a subject of interest in recent years. In both early reports of terbinafine resistance in dermatophytes by Osborne et al. T. rubrum was the resistant species, L393F in the first and F397L in the second report were the point mutations that have been found as the main cause of resistance2,13. Another decade passed before other scientists started to look at this issue and comprehending this problem has been refined. To date, T. rubrum, T. interdigitale, T. indotineae, T. mentagrophytes, and T. tonsurans have been named as resistant species in several reports3–5, 9,14–18.
Although the most common causes of this resistance is the point mutation that cause L393F and F397L substitution in amino acids 3–5, 14,17,19, other mutations such as L393S 3,16,18, F397I 3, F397V 3, F415I 3, F415S 3, 18, F415V 3, 16, H440Y 3, 18, F397L A448Y 15,20, L393S A448Y 15, Q408L 21, F484Y 18, I121M V237I 18, and F377L A448Y 16 have been reported as well. Shankarnarayan et al. reported the presence of the mutation (C1191A) among T. mentagrophytes complex isolates responsible for causing dermatophytosis in India. C to A transversion was responsible for amino acid substitution (phenylalanine to leucine) in 397th position of SQLE gene in terbinafine resistant isolates6.
In this study, we reported the presence of a novel mutation, T1189C, C1191A, and A1223T, in which the first two lead to F397L and the latter one leads to Q408L amino acid substitutions among our T. mentagrophytes complex isolates. In several studies, terbinafine MIC concentration for T. mentagrophytes complex strain with F397L mutation was determined in concentrations 64 µg/ml13; 3.2 and 6.4 µg/ml3; 4, 8 and > 32 µg/ml 4,19; 2, 4, 8, and 16 µg/ml6; >4, 8 and > 32 µg/ml18; >32 µg/ml15. In our study, it was determined 1 and 16 µg/ml. Hsieh et al. reported a MIC value > 1 µg/ml in Q408L mutation, but we could not find a correct evaluation because in our study it was 8 µg/ml21. Also, for the L393F mutation, as one of the main mutations reported so far, the MIC values showed the same diversity2–4, 9, 15, 17, 18. We wonder if bringing together these mutations with other factors such as TruMDR2 gene expression alteration22 and the increase in SalA gene expression23 could be the reason for this diversity. In our study, among 83 dermatophyte strains, only 5 of them have increased MIC values and were resistant. This shows a rate of 6% in all strains and 9% among the T. mentagrophytes complex. In previous studies, Yamada et al. reported a 1% 3, Khurana et al. 61% 26, Singh et al. 32% 4, Salehi et al. 2% 9, Shankarnayaran et al 15.4% 6, Taghipour et al. 3.5% 15, Rudramurthy et al. 10.3% in all strains and 15.7% in T. mentagrophytes complex strains 5, and Kong et al. 56%16.
In this study, 58% of our strains were among the ones that were isolated from tinea pedis patients, but the terbinafine resistant strains were not among them. On the other hand, 14% of our strains were isolated from tinea corporis patients from which 80% (n = 4) of our resistant strains were from this group and 20.5% of them were isolated from tinea cruris patients that included 20% (n = 1) of our resistant strains. So, all of the terbinafine resistant T. indotineae (according to the past taxonomy) were isolated from tinea corporis and tinea cruris. The same correlation between T. indotineae (according to the past taxonomy) and these two types of dermatophytosis was mentioned by Rudramurthy et al., Pashootan et al., and Khurana et al. 5,8,19. In accordance with this fact and the high GM values of these two diseases and especially tinea corporis (GM = 19), we recommend that in the face of this disease, combined therapy approaches should be used.
Our results showed that there was no correlation between MIC concentration of terbinafine, the infected part of the body, and mutation type. Also, in the evaluation of antifungal drug susceptibility of T. indotineae and T. mentagrophytes clinical isolates, itraconazole was more effective than griseofulvin (MIC50 = 0.25 compare to 1). Although there is ample evidence of combined azole and terbinafine resistance in dermatophytes, these reports are mostly about fluconazole, not itraconazole16, 20, 24.
We compared our MIC results to terbinafine with some other articles from Iran that have been published in recent years. In 2016, Afshari et al. reported MIC50 and MIC90 equal to 2 and 4 µg/ml and GM = 1.097, respectively25. In Salehi et al. these numbers sequentially were 0.03, 1.03 and 0.04 µg/ml9. Rezaei-Matehkolaei et al. reported 0.008, 0.016 and 0.009 µg/ml for MIC50, MIC90, and GM, respectively26. In our study, MIC50, MIC90, and GM were evaluated at 0.03, 0.03 and 0.02 µg/ml, respectively. Shen et al. searched for terbinafine susceptibility testing of T. rubrum, T. mentagrophytes and T. interdigitale and reported MIC range of terbinafine from < 0.001 to > 64 µg/ml, in a total of 2235 T. rubrum strains which 139 strains were resistant to terbinafine27. The reported MICs ranged from < 0.001 to > 64 mg/L. High MICs were reported particularly in isolates with SQLE gene mutation. Also, Bhatia et al. reported 0.50 µg/ml for MIC50 and 2.0 µg/ml for MIC9028. Furthermore, Kurup et al. reported MIC50 and MIC90 0.125 and > 5.0 µg/ml, respectively29. Taghipour et al. mentioned that MIC50, MIC90 and GM, for T. mentagrophytes strains were 0.625, > 32 and 0.12 µg/ml, respectively15. In our study, MIC50, MIC90, and GM were reported 0.03, 1 and 0.04 µg/ml, respectively. It seems the heterogeneity of the Trichophyton isolates, and the usage of different AFST methods are the main reason for the variation in MIC.
Based on ITS sequence, at least 10 genotypes of T. mentagrophytes and T. interdigitale are currently described by performing sequencing of the ITS region of the rDNA. These genotypes are mostly associated with the geographic origin or to the source of infection. In all reports the genotype of terbinafine -resistant T. mentagrophytes strains belonged to genotype VIII, which was recently announced as T. indotineae11 and T. interdigitale genotype II as resistant genotypes14,15. Our results support this claim and add T. mentagrophytes genotype VII to it. This strain had C1191A mutation and MIC equal to 16 µg/ml. Verma et al. noticed the epidemic-like situation of dermatophytosis in India and reported the emergence of a new Indian genotype T. mentagrophytes ITS type VIII30. In addition, Taghipour et al. suggested this genotype (T. mentagrophytes genotype VIII) can develop antifungal resistance31. Furthermore, this genotype was known to be of Indian origin and also was reported in countries like Iran, Oman, and Australia 17,30,31. Regarding the fact that articles with a high percentage of terbinafine resistant strains were all from India 4–6, 19 or their resistant strains originated from this country16, moreover, the features discussed above in two studies and another similar study32, we assumed that this high percentage in mentioned reports are all owing to this new emerging species, T. indotineae.