Correlation of In Vitro Susceptibility Based on MICs and Squalene Epoxidase Mutations with Clinical Response to Terbinafine in Patients with Tinea Corporis/Cruris

Recalcitrant dermatophytoses are on the rise in India. High MICs of terbinafine (TRB) and squalene epoxidase (SQLE) gene mutations conferring resistance in Trichophyton spp.

ABSTRACT

Recalcitrant dermatophytoses are on the rise in India. High MICs of terbinafine (TRB) and squalene epoxidase (SQLE) gene mutations conferring resistance in Trichophyton spp. have been recently documented. However, studies correlating laboratory data with clinical response to TRB in tinea corporis/cruris are lacking. For this study, we investigated the clinicomycological profile of 85 tinea corporis/cruris patients and performed antifungal susceptibility testing by CLSI microbroth dilution and SQLE mutation analysis of the isolates obtained and correlated these with the responses to TRB. Patients confirmed by potassium hydroxide (KOH) mounting of skin scrapings were started on TRB at 250 mg once a day (OD). If >50% clinical clearance was achieved by 3 weeks, the same dose was continued (group 1). If response was <50%, the dose was increased to 250 mg twice a day (BD) (group 2). If the response still remained below 50% after 3 weeks of BD, the patients were treated with itraconazole (ITR; group 3). Overall, skin scrapings from 64 (75.3%) patients yielded growth on culture. Strikingly, all isolates were confirmed to be Trichophyton interdigitale isolates by internal transcribed spacer (ITS) sequencing. Thirty-nine (61%) of the isolates had TRB MICs of ≥1 µg/ml. Complete follow-up data were available for 30 culture-positive patients. A highly significant difference in modal MICs to TRB among the three treatment response groups was noted (P = 0.009). Interestingly, 8 of the 9 patients in group 3 harbored isolates exhibiting elevated TRB MICs (8 to 32 µg/ml) and SQLE mutations. The odds of achieving cure with TRB MIC < 1 µg/ml strains were 2.5 times the odds of achieving cure with the strain exhibiting MIC ≥1 µg/ml.

INTRODUCTION

Tinea corporis/cruris has been successfully managed with terbinafine (TRB) in the past. Although the standard treatment recommendation for tinea corporis/cruris is TRB 250 mg once a day (OD) for 2 to 3 weeks, even lesser treatment durations have been reported to be successful (1–3). Recently, however, a number of reports have documented clinical failure of TRB treatment (4–9). High MICs and mutations in the target enzyme, i.e., squalene epoxidase (SQLE), have also been demonstrated among Trichophyton spp. in recent reports (8–12). Consequently, the use of high TRB dosages, in treatment of dermatophytoses, is becoming common (6, 13). However, it is important to emphasize that clinical breakpoints have not been defined for TRB in dermatophytoses and that the in vitro data cannot be directly applied to clinical situations. In the present study, we investigated the clinicomycological profile of patients with tinea corporis/cruris and correlated the clinical response achieved using the standard treatment and higher dose/durations of TRB with the MICs obtained by the Clinical and Laboratory Standards Institute broth microdilution method (CLSI-BMD) and with target gene (SQLE) mutations. We also analyzed the response to itraconazole (ITR) of patients in whom TRB treatment failed.

RESULTS

Among samples from 85 patients investigated in the present study, 64 (75.3%) yielded growth on culture and were further analyzed. Of those 64 patients, 44 were males and 20 were females. The ages of the patients ranged from 14 to 71 years, with a mean age of 34.9 years. Disease duration ranged between 3 weeks and 5 years, with an average duration of 8.8 months. Thirty-five (54.6%) patients had a positive family history of dermatophytic infections. Notably, 45 patients (70%) gave a history of sharing of towels between family members, highlighting the importance of fomite transmission in households. Interestingly, only two patients had pets at home and those were apparently uninfected per the accounts of the patients. Eight patients were involved in regular outdoor sports activities, and 37 gave history of prolonged working under hot and humid conditions, either at home or as a part of their occupation.

Most patients (n = 51) scored their pruritus at between 8 to 10, on a scale of 0 to 10. Forty-five (70.3%) patients had a history of application of topical steroid creams, either as a plain steroid or as a combination with antifungals. The most commonly used topical steroid was clobetasol (n = 27) followed by betamethasone (n = 17), beclomethasone (n = 5), fluticasone (n = 2), and mometasone (n = 2). Seven patients had used two or more classes of topical steroids. Interestingly, 71% (n = 32) of patients had procured steroid and or antifungal creams over the counter from pharmacists without a prescription, while the remaining 28.8% (n = 13) had been given prescriptions by general practitioners. Nine patients had apparent signs of topical steroid abuse in the form of striae, skin thinning, and depigmentation. Three of the patients had type II diabetes mellitus and were on treatment, while one each had coronary artery disease and iron deficiency anemia.

Most patients (n = 30; 46.8%) had 5 to 10 lesions at presentation, while 16 (25%) had 10 to 20, 13 (20.3%) had more than 20, and 5 (7.8%) had 5 or fewer lesions. Notably, 59.3% (n = 38) of the patients had large confluent lesions covering extensive areas. Associated inflammation was moderate in 53% (n = 34) cases on clinical examination, mild in 31.2% (n = 20) cases, and severe with pustulation in three (4.6%) cases. About 11% (n = 7) of the patients had a mixed picture, with various degrees of inflammation at different body sites.

Trichophyton interdigitale was isolated from 64 patients. The MICs for TRB, ITR, fluconazole (FLU), voriconazole (VRC), ketoconazole (KTC), amphotericin B (AMB), GRIS, miconazole (MCZ), clotrimazole (CLT), luliconazole (LUZ), and sertaconazole (SER) for 64 T. interdigitale isolates are tabulated in Table 1. Luliconazole had the lowest MICs (MIC₉₀, 0.007 µg/ml) while the MICs for fluconazole were consistently high (MIC₉₀, 32 µg/ml). Thirty-nine (61%) isolates had MIC of ≥1 µg/ml to TRB, and 24 (37%) had MIC of 0.5 µg/ml, whereas only 1 had MIC of 0.25 µg/ml, the lowest MIC recorded in T. interdigitale isolates obtained from patients.

TABLE 1.

MICs of the 64 isolates to the 11 antifungals tested^a

Species (no. of isolates)	Parameter	Drug (µg/ml)
		TRB	ITR	FLU	VRC	KTC	AMB	GRIS	MCZ	CLT	LUZ	SER
T. interdigitale (64)	GM	2.813	0.287	13.205	0.202	0.666	0.380	2.874	1.978	1.836	0.005	0.842
	MIC50	1	0.25	16	0.25	0.5	0.5	2	2	2	0.007	1
	MIC90	32	1	32	0.5	0.5	0.5	4	4	4	0.007	2
	Range	0.25 to ≥32	0.06 to ≥16	0.5 to ≥64	0.06 to 2	0.25 to ≥32	0.25 to 1	0.5 to ≥8	0.25 to ≥16	0.5 to 8	0.0035 to 0.125	0.25 to ≥16
Reference strains
T. mentagrophytes ATCC MYA-4439	MIC range	0.002 to 0.008	0.06 to 0.12	16	0.06 to 0.12	0.25 to 0.5	0.5	0.25 to 0.5	1 to 2	0.25 to 0.5	0.0035 to 0.007	0.5
T. rubrum CBS 592.68	MIC range	0.5 to 1	0.06 to 0.125	0.5 to 2	0.015 to 0.125	0.25 to 0.5	0.5	0.5 to 1	1	1	0.0035	0.5 to 1
C. parapsilosis ATCC 22019	MIC range	0.5 to 1	0.06 to 0.5	1 to 4	0.03 to 0.25	0.06 to 0.25	0.5 to 2	4 to >8	1 to 2	0.06 to 0.125	0.25	0.03 to 0.25
C. krusei ATCC 6258	MIC range	4 to ≥32	0.25 to 1	16 to ≥64	0.12 to 0.5	0.25 to 1	1 to 4	≥8	0.5 to 2	0.125 to 0.5	0.03 to 0.5	0.125 to ≥16

^aGM, geometric mean; TRB, terbinafine; ITR, itraconazole; FLU, fluconazole; VRC, voriconazole; KTC, ketoconazole; AMB, amphotericin B; GRIS, griseofulvin; MCZ, miconazole; CLT, clotrimazole; LUZ, luliconazole; SER, sertaconazole.

Among the culture-positive patients, complete follow-up data were available for 30 patients, and these were further included for the clinicomycological correlation (Table 2). Notably, 50% (n = 15) responded to OD TRB therapy (group 1). However, of these 15 patients, only two responded within the recommended 3 weeks of treatment, while the remaining 13 patients needed longer durations to respond. The mean duration of treatment given to obtain complete cure in group 1 was 39.46 ± 13.12 days (range, 21 to 66 days), and the geometric mean (GM) MIC of TRB in these 15 patients was 1.515 µg/ml. The other 15 patients did not achieve >50% clearance in skin lesions with OD TRB until 3 weeks and were shifted to the same dose administered twice a day (BD). Six of these showed >50% clearance of skin lesions after 3 weeks of updosing and were continued on this dose until complete cure (group 2). The GM MIC for group 2 was 5.039 µg/ml, and the average duration of treatment (including OD and BD treatment) was 55.66 ± 20.48 days (range, 42 to 86 days). Finally, nine patients (group 3) did not achieve 50% clinical clearance even after 6 weeks of TRB treatment and were treated with itraconazole (ITR) 100 mg BD for durations ranging from 21 to 51 days (mean, 31.88 ± 3.64 days). The GM MIC of TRB in group 3 was 14.814 µg/ml. An ITR GM MIC of 0.31 µg/ml was recorded in these 9 patients, and the MIC values ranged from 0.125 to 1 µg/ml.

TABLE 2.

Comparison of clinical response and mycological data of 30 patients^a

Patient no.	TRB response group	Previous TRB exposure	Duration of treatment with TRB in groups 1and 2 or ITR in group 3 (no. of days)	TRB MIC (µg/ml)	Amino acid substitution in SQLE protein
1	3	O	ITR (28)	≥32	F397L
2	3	T	ITR (21)	≥32	L393F
3	3	O and T	ITR (45)	≥32	F397L
4	3	T	ITR (36)	≥32	F397L
5	3	—	ITR (36)	≥32	L393F
6	3	—	ITR (51)	8	F397L
7	3	O	ITR (28)	≥32	F397L
8	3	T	ITR (21)	≥32	F397L
9	3	O and T	ITR (21)	0.5	ND
10	2	O	TRB (86)	1	¶b
11	2	—	TRB (42)	1	¶
12	2	—	TRB (42)	≥32	F397L
13	2	—	TRB (54)	≥32	F397L
14	2	—	TRB (35)	≥32	ND
15	2	—	TRB (75)	0.5	ND
16	1	—	TRB (35)	1	¶
17	1	—	TRB (21)	1	ND
18	1	—	TRB (52)	1	ND
19	1	—	TRB (42)	1	¶
20	1	—	TRB (39)	1	ND
21	1	—	TRB (28)	1	ND
22	1	—	TRB (43)	1	ND
23	1	—	TRB (66)	0.5	ND
24	1	—	TRB (35)	0.5	ND
25	1	—	TRB (28)	≥32	F397L
26	1	—	TRB (43)	≥32	L393F
27	1	—	TRB (42)	4	F397L
28	1	—	TRB (35)	2	ND
29	1	—	TRB (21)	0.5	¶
30	1	O	TRB (62)	0.5	ND

^aO, oral; T, topical; O and T, oral and topical; —, no previous exposure to terbinafine; ND not done.

^b¶, negative for substitution in SQLE.

The difference among the three groups with respect to the GM MICs of TRB was highly significant (P = 0.009). Also, there was a statistically significant difference between the GM MIC of combined group 1 and 2 and that of group 3 (P = 0.01). No statistically significant correlation was noted between individual MICs (based on MIC ≥ 1 µg/ml or <1 µg/ml), with cure achieved with TRB.

In contrast to 83.3% of patients infected with strains exhibiting a TRB MIC of <1 µg/ml, 66.7% of those infected with strains exhibiting a TRB MIC of ≥1 µg/ml achieved cure, either by standard dose/duration of treatment or by updosing/increasing treatment duration. The odds of achieving cure with the strains exhibiting a TRB MIC of <1 µg/ml were 2.5 times those of achieving cure when the strain had a MIC of ≥ 1 µg/ml (Table 3). Squalene epoxidase gene mutation analysis was done in 22 of 64 isolates. These included 18 patients from the three clinicomycological correlation groups (group 1, n = 6; group 2, n = 4; group 3, n = 8). The remaining four isolates were randomly selected from patients who were harboring isolates with TRB MICs of 0.5 µg/ml. Of these, 13 harbored mutations leading to single amino acid substitution in the SQLE protein (Table 2). Ten isolates were found to have amino acid substitution Phe397Leu in SQLE protein, while three had the substitution Leu393Phe.

TABLE 3.

Response to TRB in mycologically susceptible and resistant infections

Infection status	No. (%) of infections:		Odds ratio
	Susceptible (MIC < 1 µg/ml); n = 6	Resistant (MIC > 1 µg/ml), n = 24
Cure achieved with TRB (n = 21)	5 (83.3)	16 (66.7)	2.50
Not cured with TRB (n = 9)	1 (20)	8 (32)

DISCUSSION

Dermatophytoses present a huge economic burden on the medical establishments all over the world, with an estimated worldwide prevalence of 20% to 25% (14). The emerging recalcitrant dermatophytosis has thus become a distressing medicoeconomic concern in India (4). The data presented here form the basis of establishing clinical breakpoints for a drug-species pairing, although higher patient numbers and intricate tissue-level pharmacokinetic (PK) data would also be essential for the same. A striking finding of the present study was the dominance of T. interdigitale as the etiological agent for tinea corporis/cruris. Previous worldwide studies as well as older Indian data report T. rubrum as the predominant dermatophyte causing tinea corporis/cruris, while T. interdigitale has previously been reported as a prominent species from only a few geographical areas (15–20). However, a few recent Indian reports have also found T. interdigitale in a large percentage of isolates (9, 10, 21). Our results also reconfirm the unfortunate trend of high MICs of TRB for T. interdigitale. No epidemiologic cutoffs (ECVs) have been established for TRB resistance in T. interdigitale by CLSI. The previously reported TRB MIC cutoff value of ≥1 µg/ml for T. rubrum strains (22) was applied for TRB resistance in T. interdigitale isolates investigated in the present study. Notably, highly variable MICs (0.001 to 32 µg/ml) of TRB have been previously published for clinical isolates of T. interdigitale and T. mentagrophytes (10, 18). Further, several studies have reported terbinafine MICs for T. interdigitale wild-type strains to be much lower (≤ 0.06 µg/ml) than the MICs observed in the present study (lowest MIC, 0.25 µg/ml) (23–31). However, it is important to emphasize here that the antifungal susceptibility testing (AFST) of dermatophytes by CLSI is still not validated and that the reproducibility of MIC data in multicenter studies is warranted. Further, due to the slow-growing nature of dermatophytes and to contamination, resistance determination is often challenging (32). Therefore, it is crucial that the criterion of high TRB MICs of ≥1 µg/ml against T. interdigitale be validated by multicenter studies with large number of geographically diverse isolates using the defined reference broth microdilution method. Among other systemic antifungals, only one isolate was resistant to ITR (MIC of ≥8 µg/ml), one to VRC (MIC of ≥2 µg/ml), and three to ketoconazole (MIC of ≥8 µg/ml) (33).

It was interesting that only two patients (patients 17 and 29) responded to the conventional dose and duration of TRB. The remaining patients in group 1 required much longer courses ranging from 28 to 66 days. Extrapolating from the data on azoles, it is known that optimizing drug exposures can improve clinical outcomes in the context of raised MICs/resistant strains, depending on variables such as elevated MICs, resistance mechanisms, and pharmacokinetics/pharmacodynamics (PK/PD) properties of the antifungal agent (34). For drugs used against dermatophytoses, the levels in tissue (stratum corneum [SC]) are more important than the plasma levels. Prolonged courses have been shown to achieve higher levels of TRB in the SC, on the basis of previously reported PK studies (35, 36). The higher levels thus achieved with longer durations may have been able to offset the higher MICs in group 1 patients.

Further, on updosing to BD, another six patients achieved complete cure. TRB has a linear PK profile of up to 750 mg of dose, implying that an increase in dose to this level would increase plasma levels proportionately (37). This would in all likelihood lead to an increased SC level, unless the uptake by skin is saturable; there is no documentation of that yet to the best of our knowledge. The group 2 patients thus benefitted from both an increase in dose and an increase in duration, possibly achieving higher SC levels than were achieved by group 1. Notably, the GM MIC of this group (5.039 µg/ml) was about five times higher than that of group 1 (1.515 µg/ml). This may explain why the group 2 patients did not respond only with longer durations of treatment.

Whether doses higher than those that we used in the present study would improve cure rates further would require further testing in clinical trials, though drug toxicity may become a limiting factor. In the present study, drug-related adverse effects were not observed with longer durations and higher doses of TRB, although the data may be biased due to the small sample size investigated (38).

Clinical response data were available for all 13 patients harboring T. interdigitale isolates carrying mutations in the SQLE gene. Eight (61.5%) of these did not respond to higher drug (TRB) exposure levels, while five (38.4%) responded. Thus, higher TRB exposure could offset the effect of SQLE mutations in many patients harboring mutated strains. However, this interpretation warrants further analysis in a large set of patient population data that include TRB responders and nonresponders. In both the present study and our previous work, SQLE mutations were noted in isolates with TRB MICs ranging 4 to ≥32 μg/ml and a wild-type genotype (i.e., no SQLE mutation) was noted in T. interdigitale isolates with MICs of ≤2 μg/ml. This may imply that SQLE mutations lead to high-level resistance in T. interdigitale and that alternate mechanisms may be working in resistant isolates with MICs of <4 μg/ml (9).

We could not identify any host factors predisposing to TRB resistance. Topical steroid use may enhance development of resistance to antifungals being used simultaneously by activating fungal metabolism and by cell membrane-protective activity. However, we did not find a statistically significant correlation between topical steroid use and clinical response to TRB. One possible reason for this could have been the widely prevalent use of topical steroids in our patients, which precluded formation of comparable groups. In the present study, prior TRB exposure did not yield any predictable results, although we observed previous TRB exposure to be most prominent in group 3 (77.78% in group 3 versus 6.67% in group 1 and 16.67% in group 2). We also did not find a significant correlation between clinical response to TRB and individual MICs. This can probably be attributed to the small sample size. Larger studies may be able to clarify this aspect. Whether the resistance noted in this study was primary or acquired cannot be commented upon in a single-time assessment. But an interesting finding was that most (14/16; 87.5%) of the patients who harbored resistant isolates and yet responded to TRB had not been exposed to TRB before, while most of those who harbored resistant isolates and did not respond to TRB (6/8; 75%) had been exposed to TRB before.

To conclude, the present report highlights the worrisome level of TRB resistance in our clinical cases of tinea corporis/cruris. Increasing drug exposure by means of higher dose/duration can offset this to some degree, but the high failure rate (about 30%) cannot be ignored. A reconsideration of the treatment protocol for tinea corporis/cruris is warranted. However, it is equally important to consider the larger scenario of drug resistance among fungi while forming recommendations to treat dermatophytoses. And it is also very relevant to curtail the over-the-counter availability of antifungal and steroid creams to prevent further unfortunate consequences similar to what we are witnessing in India already.

MATERIALS AND METHODS

The study was approved by the institutional ethics committee and registered with the clinical trials registry of India (CTRI). The presented patients were recruited between July 2016 and December 2017. Eighty-five consecutive patients with tinea corporis, with or without tinea cruris, presenting to the dermatology outpatient department (OPD) of Ram Manohar Lohia hospital, Delhi, were included after informed consent was obtained. Diagnosis was made on clinical examination and confirmed by direct microscopy using a 10% KOH mount in the OPD. The included patients had at least five lesions over the glabrous skin and/or a large lesion(s) covering significant body surface area and were judged by the primary investigator (A. Khurana) to require systemic treatment. Patients who had used any systemic antifungal in the preceding 4 weeks or had used any topical antifungal or steroid in the preceding 2 weeks were excluded. Pregnant or lactating women and children less than 12 years of age and/or weighing less than 40 kg were also excluded. Details of disease onset, duration, and course; family history; and previous treatments were recorded followed by examination of the entire skin surface. Photographs were taken for documentation and comparison of treatment responses (PowerShot G12 camera [Canon, New York, USA]). Skin scales were collected and transported to the Medical Mycology Laboratory of Vallabhbhai Patel Chest Institute, Delhi, in a thick dry sheet of paper. Treatment was started upon direct KOH mount confirmation and after sending the samples for culture. Each patient was treated until the patient achieved complete cure, which was defined as complete clinical clearance along with a negative KOH mount result from the site of initial sampling. For the purpose of clinicomycological correlation, three TRB response groups were formed as described below.

The patients were started on TRB 250 mg OD and asked to report for follow-up visits every 3 weeks. The treatment response was evaluated by the primary investigator at each visit by clinical examination and comparison with previous photographs. If the response to this regimen was greater than 50% at 3 weeks follow-up, the same dose was continued until complete cure. This group of patients was labeled group 1. If the clinical response after 3 weeks was less than 50% or if new lesions appeared during this time, the patient’s treatment was changed to TRB 250 mg BD. As 3 weeks has previously been recommended as the standard duration of treatment of tinea corporis/cruris with TRB 250 mg OD, this cutoff was chosen for shifting to a higher TRB dose (1, 39). If >50% clinical clearance was achieved within 3 weeks, the same regimen was continued until complete cure and patients were assigned to group 2. However, if the response still remained below 50% or new lesions appeared, TRB was stopped and the patients were administered ITR in a dose of 100 mg BD and treated until complete cure (group 3). The patients were counseled on general hygiene measures and conduct to reduce transmission among family members. To avoid additive effects, no topical antifungal was given. Information on any adverse effects pertaining to TRB was recorded at each visit, and liver function tests were performed periodically.

Skin scrapings were processed for direct microscopic examination by the use of 10% potassium hydroxide (KOH)/Blankophor mounts. The specimens were inoculated on two sets of media each, one consisting of Sabouraud’s dextrose agar (SDA) containing gentamicin and chloramphenicol and the other of SDA containing cycloheximide (0.05%), and were incubated at 28°C for 2 weeks. The primary culture on SDA was transferred to potato dextrose agar (PDA) and incubated at 28°C for 14 days for macroscopic phenotypic identification. For analysis of microscopic morphological details, slide cultures were prepared on PDA and mounted in lactophenol cotton blue mount.

Mycological investigations.

Molecular identification of all the 64 isolates was performed by sequencing the internal transcribed spacer (ITS) region of the small subunit ribosomal DNA (rDNA) (9, 40). ITS sequences were subjected to BLAST searches at GenBank (https://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). Sequence-based species identification was defined by ≥99% sequence similarity with ≥99% query coverage. The neotype and type strains of Trichophyton species (T. interdigitale, CBS 428.63NT; T. mentagrophytes, IHEM 4268; T. rubrum, CBS 392.58NT; T. tonsurans, CBS 496.48NT; T. violaceum, CBS 374.92T147) were retrieved from GenBank and included for phylogenetic analysis.

Antifungal susceptibility testing (AFST) was carried out using the CLSI-BMD M38-A2 guidelines (23). Eleven systemically/topically used antifungals were tested, including terbinafine (TRB) (R-1012/16; Synergene India, Hyderabad, India), itraconazole (ITR) (ITFP07008; Lee Pharma, Hyderabad, India), voriconazole (VRC) (030M7505V; Sigma-Aldrich, Steinheim, Germany), fluconazole (FLU) (036M4709V; Sigma-Aldrich, Steinheim, Germany), sertaconazole (SER) (OP-SANP/05/16/011; Optimus, Hyderabad, India), luliconazole (LUZ) (2823343; Sun, Baddi, HP India), clotrimazole (CLT) (075K1032; Sigma-Aldrich, Steinheim, Germany), miconazole (MCZ) (BCBD5966V; Sigma-Aldrich, Steinheim, Germany), ketoconazole (KTC) (SLBR1290V; Sigma-Aldrich, Steinheim, Germany), amphotericin B (AMB) (121K4042; Sigma-Aldrich, Steinheim, Germany), and griseofulvin (GRE) (MKBQ4861V; Sigma-Aldrich, Steinheim, Germany). CLSI-recommended control strains of Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were included for every batch of isolates tested each day. Reference strains of T. mentagrophytes (ATCC MYA-4439) and T. rubrum (CBS 592.68) were also included in susceptibility testing. MIC endpoints for all the drugs except AMB were defined as the lowest concentration that inhibited 80% of the growth as read visually at 72 h. For AMB treatment, complete inhibition of growth was taken as the MIC. CLSI document M38-A2 suggests determining MICs of dermatophytes at 4 days (96 h). However, confluent growth was observed after 3 days (72 h) of incubation for all T. interdigitale isolates identified in the present study. Therefore, MICs of all T. interdigitale isolates tested were read visually at 72 h at 35^° C to reduce the reporting time of susceptibility results. The MIC ranges for the quality control (QC) strain T. mentagrophytes (ATCC MYA-4439) against griseofulvin, itraconazole, voriconazole, and terbinafine were interpreted per CLSI M38 A2 E (23). For AMB, a breakpoint of >1 µg/ml was considered (41). MIC ranges for the QC strain T. mentagrophytes (ATCC MYA-4439) against FLU, KTC, MCZ, CLT, LUZ, and SER have not been given by CLSI; therefore, reference strain MIC ranges of these antifungals could not be interpreted.

The amplification primers for the SQLE gene were used as described previously (8). PCR was carried out in a 50-μl reaction volume, and the conditions included initial denaturation for 5 min at 95°C followed by 34 cycles of 30 s at 95°C, 30 s at 60°C, and 180 s at 72°C. DNA sequencing was performed using the PCR primers at a concentration of 2.5 mmol/liter. All sequencing reactions were carried out in a 10-μl reaction volume using BigDye Terminator kit v3.1 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommendations and were analyzed on an ABI3130xL Genetic Analyzer (Applied Biosystems). The amino acid sequences of SQLEp of all the investigated T. interdigitale isolates in the present study were compared with the reference sequence of T. interdigitale (GenBank accession number EZF33561).

Statistical analysis was done on SPSS software version 21. One-way analysis of variance (ANOVA) was used to compare the geometric mean (GM) MICs among the three treatment response groups. The chi-square test was used to compare treatment response data with demographic data.