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Abstract
Background: Dermatophytosis have assumed epidemic proportions in India. Antifungal drug resistance solely cannot explain disease magnitude and changing epidemiology. Objectives: Aim of this study was to analyse clinical-mycological aspects of dermatophytosis, and estimate contribution of drug resistance in clinical recalcitrance. Methods: This single-centre observational, cross-sectional, descriptive study was done in tertiary centre of western India after ethical approval, enrolling dermatophytosis patients of all ages and sex. After history and examination, KOH mount and culture in modified SDA medium was done. Culture positive isolates were subjected to E-strip antifungal susceptibility method to test MIC for Terbinafine, Itraconazole, Fluconazole and Griseofulvin. Results: Total 300 patients were included, with mean age of 33.83±27.5 years and male-to-female ratio of 1.22:1; tinea corporis et cruris being commonest, 39.33% (n=118). Only 11.67% (n=35) were treatment naïve, having classical annular morphology. History of topical steroid abuse was found in 81.67% (n=245), with pseudoimbricate lesions in 70.61% (n=173). 86.67% (n=260) had KOH positivity while 83.33% (n=250) had culture positivity: Trichophyton mentagrophytes 45.6% (n=114), followed by Trichophyton rubrum in 34.4% (n=86). A total of 265 patients fit into definition of recalcitrance, from which 12.45%, i.e., 33 isolates showed in-vitro fluconazole resistance. 14.33% (n=43) cases were chronic, 37% (n=111) persistent, 46% (n=138) recurrent while 17% (n=51) had relapse in their disease course. Steroid abuse was the commonest denominator. Conclusion: Role of antifungal resistance in recalcitrant dermatophytosis remains debatable. Stopping steroid abuse, which is often the commonest culprit, with adherence to standard antifungal therapy remains the paradigm in management.
Keywords: Antifungal resistance, dermatophytosis, E-test, T. mentagrophytes
Introduction 
There has been a surge in recalcitrant dermatophytosis in India and this problem is further compounded by the paucity of clinical-mycological studies. The relative contribution of antifungal drug resistance to clinical recalcitrance remains unclear. The present study was done in the context of this lacuna, with an aim to (i) estimate the prevalence, contributory factors; clinical and mycological profile of dermatophytosis; and (ii) estimate the proportion of recalcitrance caused by antifungal drug resistance.
Materials and Methods The study was conducted in the outpatient department of Dermatology in a single tertiary care center in western India with prior ethics approval. This was an observational, cross-sectional, and descriptive study conducted from August 2019 to July 2021.
Patients of all ages and sex with clinically diagnosed dermatophyte infection of skin, hair and/or nail and who gave written consent/assent for diagnostic tests, follow-up, and photography were included. Patients unwilling to the study and those with incomplete data were excluded. A total of 300 consecutive patients fulfilling the criteria were included in this study.
Basic demographic data was collected. The comorbidities and family history were noted. Morphology was analyzed on clinical grounds. The clinical course of the disease was noted with emphasis on recalcitrance, as per definition consensus by ECTODERM.[1] The duration of their fungal infection, previous treatment/s taken, and the source of their prescription was documented with pertinent details.
For mycological examination, a blunt curette and a scalpel were used to obtain the appropriate specimen, depending upon the type of dermatophytosis. It was later subjected to an appropriate concentration of 10%–40% potassium hydroxide (KOH) and mounted on a glass slide to view fungal hyphae under the light microscope. The sample was then subjected to fungal culture in modified saboraud dextrose agar media for 10–28 days of incubation under the ideal environment. The colony growth was observed for its macroscopic characteristics, color, and texture of colonies on forward and reverse slants. Microscopic examination by Lactophenol Blue mount was done to identify the different species of trichophyton, microsporum, and epidermophyton; along with any other organism growing in the sample.
The culture-positive samples were then subjected to E-strip antifungal susceptibility method to test in vitro minimum inhibitory concentration (MIC) values for the four oral antifungal drugs: terbinafine, itraconazole, fluconazole, and griseofulvin. Antifungal susceptibility was done on plates with Mueller–Hinton Agar + 2% Glucose + 0.5 mcg/ml methylene blue dye, i.e. Mueller–Hinton Agar, Modified (as per Clinical and Laboratory Standards Institute (CLSI) for antifungal) (M1825). Ezy MIC™ strip (E-test) was applied with concentrations of antifungals in E-strip as 0.016–256 μg/mL for fluconazole and 0.002–32 μg/mL for itraconazole, terbinafine, and griseofulvin each. The MIC was read where the ellipse intersected the MIC scale on the strip.
Data was analyzed using the Statistical Package for Social Sciences Version 15.0 (Chicago, IL, USA). Qualitative variables were presented as frequency and percentages. Quantitative variables were presented as mean (standard deviation) or median (range) depending upon data distribution.
Results Demographic analysis
A total of 300 patients of clinically diagnosed dermatophytes were included in this clinic-mycological study from 813 consecutive outpatients, with a prevalence of 36.9%. The mean age group of patients was 33.83 ± 27.5 years with a range of 3–65 years. The majority of the patients, 32.67% (n = 98) belonged to the age group of 21–30 years. Fifty-five percent (n = 165) were males while 45% (n = 135) were females with a male-to-female ratio of 1.22:1. Applying the modified Kuppuswami scale, 43.33% (n = 136) belonged to lower socio-economic class. The majority of patients were homemakers, 35.67% (n = 107). Twent-two percent of patients (n = 66) had comorbidities of which 19.69% (n = 13) were on systemic immunosuppressive medications. The most common comorbidities were diabetes mellitus and hypothyroidism in 2.67% (n = 8) patients each. Family history was positive in 79% (n = 237) of patients.
Clinical analysis
From the total of 300 samples, 90.67% (n = 272) were from the skin, 4% (n = 12) from hair, and 5.33% (n = 16) from nails. The most common pattern of cutaneous affliction was Tinea corporis et cruris in 39.33% (n = 118), followed by Tinea cruris in 20% (n = 60), Tinea corporis in 9.93% (n = 28), Tinea corporis et cruris et faciei in 8% (n = 24), Tinea faciei in 6.67% (n = 20), Tinea unguium in 5.33% (n = 16), Tinea capitis in 4% (n = 12), Tinea corporis et faciei in 3% (n = 9), Erythroderma in 2.67% (n = 8), and Glabrous Tinea capitis in 1.07% (n = 5) patients.
Extensive dermatophyte infection (lesions involving >20% body surface area or ≥two non-contiguous sites) was present in 54.67% (n = 164) cases. The average duration for which the patient reported to have the present disease was 3.92 ± 3.38 months, with a range of 0.5–36 months.
Among the 12 cases of Tinea capitis, 41.67% (n = 5) had a gray patch, 41.67% (n = 5) had black dot and kerion in 16.66% (n = 2) cases. Among the 16 cases of nail involvement, 56.25% (n = 9) had distal and lateral onychomycosis, 37.5% (n = 6) had total dystrophic onychomycosis while 6.25% (n = 1) had proximal subungual onychomycosis.
Treatment analysis
88.33% (n = 265) of patients gave the history of prior treatment taken, while only 11.67% (n = 35) were treatment naïve who had classical annular morphology. Over the counter medicines were opted by 74.34% of patients (n = 197). These included topical keratolytics, super potent steroids like clobetasol propionate in combination with antifungals and topical irritating agents. Combinations of potent and super potent steroids with antifungals were most common with erratic frequencies of application. 20.75% (n = 55) patients opted for treatments from local medical practitioners, 15.85% (n = 42) patients from ayurvedic remedies while 7.55% (n = 20) used some homeopathic medicines. 13.21% of patients (n = 35) also experimented with household remedies like applying garlic, paste, cement, lime, hot cooking oils, etc., over the lesions.
Prior use of topical and/or systemic antifungal agents was present in 72.33% (n = 217) of patients, while 81.67% (n = 245) had a history of using steroids from which 70.61% (n = 173) had pseudoimbricate morphology [Figure 1]. 22.87% (n = 56) patients also had a history of taking oral steroids while 21.22% (n = 52) patients had taken some injectable steroids via intralesional, intramuscular, or intravenous routes, having developed the Cushingoid stigmata and low fasting serum cortisol levels.
Figure 1: Chart showing the distribution of morphologies of steroid-modified Tinea. Pseudoimbricate pattern was seen in most patientsMycological analysis
From the total of 300 patients, 86.67% (n = 260) had KOH positivity while 83.33% (n = 250) had culture positivity. The positive predictive value of detecting the disease is 86.67% for KOH mount and 83.33% for culture. Discordance between KOH and culture results was seen in 17.33% (n = 52) cases. 8.4% (n = 21) were negative for KOH but positive for culture, while the rest 11.92% (n = 31) samples were positive for KOH but negative for culture. Considering culture to be the gold standard in terms of investigations, sensitivity, and specificity of KOH mount was 91.6% and 42%, respectively.
From the 250 culture isolates, the maximum isolates were Trichophyton mentagrophytes at 45.6% (n = 114), followed by T. rubrum at 34.4% (n = 86), T. verrucosum at 9.6% (n = 24), T. violaceum at 3.2% (n = 8), T. tonsurans at 2.4% (n = 6), Microsporum gypseum at 2.4% (n = 6), Microsporum audouinii at 0.8% (n = 2), and non-dermatophyte molds at 1.6% (n = 4) isolates. On subjecting the culture-positive isolates to drug sensitivity, high MIC values were obtained with fluconazole [Table 1].
Table 1: Median and MIC90 values of various isolates cultures in the studyA total of 33 isolates had in vitro MIC90 higher than the cut-offs as per local laboratory values, making them “resistant” to fluconazole in vitro. From these 33 resistant isolates, 69.7% (n = 23) were T. mentagrophytes, followed by 15.15% (n = 5) T. rubrum, 6.06% (n = 2) T. verrucosum, and 3.03% (n = 1) T. tonsurans, M. gypseum, and T. violaceum each.
Discussion India is experiencing an alarming increase in dermatophytosis prevalence which is reported to be as high as 36.6–78.4%.[1] Literature describes this situation as a “great Indian epidemic,” “hyperendemic,” and “haunting specter of recalcitrant dermatophytosis” which would not be an exaggeration, looking at the increase in the proportion of recalcitrant cases encountered.[2],[3] Factors amalgamating into this “daunting” picture of recalcitrance often go beyond antifungal drug resistance.[4]
[Table 2] summarizes the demography, clinical, and mycological profile of similar studies.
Table 2: Table shows the summary of the comparison between the studies done on the clinical and mycological profile of dermatophytosis in western India in the last 5 years. The disease now spans across all age groups, socio-economic strata, and largely steroid-modified pictures. The trend also shows the preponderance of T. mentagrophytes over the yearsOnly 11.76% of patients had the textbook annular morphology. This was in concordance with the 82% positivity of history of using steroids for their disease. Verma et al.[2] also had similar observations stating that the classic “annular” description of Tinea may no longer be valid in the Indian context. Corticosteroids by hampering immunity in the local milieu result in ineffective fungal elimination, which continues its centrifugal march without adequate central clearing, and thereby eventuating into bizarre morphologies and extent [Figure 2]. Similar misuse of steroids was seen by Nenoff et al.[5] where 81.3% had applied high potency steroid-antifungal-antibiotic to temporarily mitigate itch and erythema, only to end up with an atypical clinical course as the outcome. Other factors that were consistent with recalcitrance included a positive family history and lower socio-economic strata.
Figure 2: Various steroid-modified morphologies are depicted here (a) Pseudoimbricate: Multiple active borders with intermittent central clearing in a 25-year-male, due to intermittent suppression of local immunity by erratic topical corticosteroid application (b) Erythroderma: Generalized erythema and scaling in a 55-year-male using over the counter combination containing clobetasol propionate for the past 8 months. Culture revealed ample growth of T. mentagrophytesIn our study, we found plenty of hyphae in most cases denoting higher fungal load, as also seen by Poojary et al.[6] Culture yield was higher than that by Snehal et al.[7] and Kalita et al.;[8] the latter explained their low culture positivity due to contamination and inclusion of partially treated cases by antifungal agents.
Nenoff et al.[5] reported that T. mentagrophytes “Indian ITS genotype” might be disseminated Indian-wide due to widespread topical steroid abuse (now called the T. indotineae). In our study as well, we found a preponderance of T. mentagrophytes, but molecular studies could not be performed due to resource constraints. This epidemiological shift from T. rubrum to T. mentagrophytes has clinical repercussions. Zoophilic T. mentagrophytes has acclimatized and anthropized having easier transmissibility, more virulence, and better survival on fomites.
Since no CLSI standards exist for MICs of antifungals for dermatophytosis, MIC90 values from our results were compared with similar studies done over recent years [Table 2] and [Table 3]. MIC >1 μg/ml is vaguely suggested as a cut-off of in vitro resistance for systemic antifungals.[9] Based on this, rising MICs and in vitro resistance to fluconazole was seen in 12.45% (n = 33) isolates, while isolates remained sensitive to the other drugs in our study.
Table 3: Comparison of MIC90 values of T. mentagrophytes in various studies[20]Recalcitrant Tinea infection is a generic term that encompasses relapse, recurrence, re-infection, persistence of infection, and chronic infection.[1] A total of 88.3% (n = 265) patients fit into these subgroups/inclusions of “recalcitrance,” with the relative contribution of various factors depicted in [Table 4]. The median time to relapse of lesions was 9 weeks (range of 6 weeks to 6 months). Antifungal resistance was the least common contributor to clinical recalcitrance, while steroid abuse was the most common underlying factor.
Table 4: Subgroups of clinical recalcitrance in the study with their individual prevalence. Definitions have been adapted from the ECTODERM consensus by Rajgopalan et al.[1]MIC is a construct of laboratory interpretation of inhibition zones.[10] However, in practicality, multitude of agent, host, environmental, and drug factors orchestrate the clinical picture of this mycosis. This raises two important questions: how should the MIC values be interpreted in the clinical context, and whether drug resistance is actually to be blamed for clinical recalcitrance? Vandeputte et al. stated that there is “no conclusive evidence to implicate in vitro resistance with therapeutic failure” in dermatophyte infections.[11] Clinical break-point which is the antifungal concentration determining clinical success is yet to be defined for dermatophytes.[12] Hence, the need of the hour is the generation of epidemiological cut-off value for any given species and antifungal agent, which is defined as MIC value that provisionally differentiates the “wild-type isolates (generally considered as susceptible) from non-wild-types (generally considered as resistant isolates),” which can guide the selection of antifungals in a practical scenario.[13]
Limitations of this study include the use of an E-test and the lack of performing pharmacokinetic studies. Only a few studies have so far reported the use of the E-test methodology for dermatophytes, making the comparison of MIC values difficult. Recently, a good correlation between E-test results and the broth dilution method is reported in the literature.[14] The E-test method is simpler and rapid (3–4 days) compared with micro- and microbroth dilution (more than 7 days). Ideally, in vitro sensitivity should be interpreted after quantifying drug levels reaching the stratum corneum and observing the clinical response of the drugs in vivo.[10] Hence, the clinical translation of laboratory MIC values remains non-linear and imperfect.
Conclusion Antifungal resistance as a standalone factor seems to be less important in the present scenario of dermatophytosis. While values of MIC get more clarification in the sense of in vivo–in vitro concordance; curbing steroids, general measures on fomites along with standard antifungal therapeutic regimen should be the standard of care.
Declaration of patient consent
Written informed consent was obtained from all individual participants included in the study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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