Abstract
Background:
The currently reported prevalence of dermatophytosis (superficial mycoses) in India ranges from 6.09%–61.5%. Due to the rising cases of treatment failure, chronic recalcitrant disease, and frequent relapses, standardization of antifungal susceptibility testing of dermatophytes has become important.
Aims and Objectives:
To determine the in vitro susceptibility profile of different isolates of dermatophytes to eight antifungal drugs by broth microdilution method.
Materials and Methods:
In total, 236 consecutive patients who were clinically suspected of suffering from dermatophytosis were included in this cross-sectional descriptive study. Nails, hair clippings, and skin scrapings were tested by the KOH mount and cultured on Sabouraud’s agar medium (incubated at 25°C and 37°C) and dermatophyte test media. The dermatophytes isolated were identified based on the morphology of the colony growth as observed in the lacto phenol cotton blue (LPCB) mount, temperature tolerance, urease production, and in vitro hair perforation test. Antifungal susceptibility test was performed for dermatophytes against eight antifungal agents (terbinafine, griseofulvin, itraconazole, fluconazole, voriconazole, ketoconazole, luliconazole, and posaconazole) using the Clinical and Laboratory Standards Institute broth microdilution method (M38-3rd edition).
Results:
Trichophyton rubrum was the most common dermatophyte species isolated in the study. Triazoles were found to have low minimum inhibitory concentration (MIC), except fluconazole, which showed very high MIC values with 55.2% isolates showing resistance (MIC ≥ 32 μg/mL). Luliconazole had the lowest MIC90 (0.003 μg/mL). There was a wide variation in the MIC values for terbinafine (0.06–16 μg/mL) and griseofulvin (0.125–8 μg/mL) with 13.1% and 52.6% of isolates showing resistance (MIC ≥ 4 μg/mL) to terbinafine and griseofulvin respectively.
Conclusion:
The present study showcases the MIC values of eight anti-fungal drugs used for the treatment of different species of dermatophytes. It was observed that the commonly used antifungals such as fluconazole, griseofulvin, and terbinafine showed high MIC values compared to newer drugs such as luliconazole, posaconazole and voriconazole. More studies based on species distribution and antifungal susceptibility testing (AFST) should be performed to understand the changing epidemiology of dermatophytosis.
KEY WORDS: Antifungal susceptibility test, dermatophytosis, luliconazole, minimum inhibitory concentration, trichophyton
Introduction
The prevalence rate of superficial mycotic infection has been estimated by the World Health Organization (WHO) to be 20–25% globally.[1] The most important and common superficial fungal infection is dermatophytosis caused by dermatophytes, a group of fungi that can grow by invading the keratin of the skin, hair, and nails. Based on the natural habitat, dermatophytes are differentiated into geophilic, zoophilic, and anthropophilic species.[2,3] Dermatophytosis can lead to poor quality of life when not managed properly. Risk factors include inappropriate treatments with steroid combination creams, improper dosages of antifungals, and unhygienic lifestyle.[4,5,6]
The surge in treatment failure, chronic recalcitrant diseases and frequent relapses necessitate the standardization of antifungal susceptibility testing (AFST) of dermatophytes. This will generate data on in vitro minimum inhibitory concentration (MIC) values of currently available and newer drugs for improvement in the treatment plan of dermatophytosis. The current study aims at generating and analysing in vitro MIC data for different isolates of dermatophytes against eight antifungal drugs using broth microdilution.
Aims and Objectives
To determine and compare the in vitro susceptibility profile of different isolates of dermatophytes to eight antifungal drugs by the broth microdilution method.
Materials and Methods
A total of 236 clinically diagnosed cases of dermatophytosis were included in this cross-sectional study. The study was conducted over 1 year in a tertiary health care centre after institutional ethics committee approval (IEC/2020/66). The demographic details, relevant history and clinical examination of cutaneous lesions were recorded.
Inclusion criteria: All clinically diagnosed patients of dermatophytosis, that is, mycotic infections localised to superficial layers of skin, hair and nails irrespective of age and sex.
Exclusion criteria: Any patients of dermatophytosis who were taking or had taken anti-fungal treatment during the last 3 months.
Mycology laboratory procedures
Skin scrapings, hair and nail clipping were dissolved in 20% potassium hydroxide (KOH) for the digestion of keratin and non-fungal elements. All specimens were inoculated on Sabouraud’s dextrose agar (SDA) with chloramphenicol, gentamicin and cycloheximide and dermatophyte test medium (DTM), incubated at 25°C and 37°C for 4 weeks. For species identification of dermatophyte isolates tests such as lacto phenol cotton blue (LPCB) mount, calcofluor white (CFW), slide culture, urease and hair perforation tests were conducted. Each culture-positive sample was observed for gross morphology, mycelial type, and conidial arrangement (macro and microconidia) to differentiate between species and genera.[7]
Antifungal Susceptibility Testing (AFST)
AFST was conducted for dermatophytes isolated from patients’ specimens by the broth microdilution method (BMD) as per the CLSI-M38 3rd ed.[8] All drugs were from Sigma Aldrich except ketoconazole (KTC){HIMEDIA} and luliconazole (LULI){Optrix Lab Pvt Ltd. India}. Homogenous suspensions of the supernatant were collected in new sterile tubes and adjusted to 0.11 O.D. at 530 nm by a densitometer. The concentration of the suspension was adjusted according to the spore count. The stock solution of antifungal drugs was prepared according to the standard protocol. Stock solution of all drugs was prepared in dimethyl sulfoxide, at a concentration of 2 mg/mL (except FLC, which was dissolved in water). Ten two-fold drug dilutions were prepared in the RPMI medium from stock solutions in a 96-well microtiter plate to assess the MIC of antifungal drugs. Then, 5 mL of the stock solution was prepared from powder and drugs dissolved.The concentrations tested for KTC (0.015–8 μg/mL), itraconazole (ITC){0.03–16 μg/mL}, voriconazole (VRC){0.001–0.5 μg/mL}, griseofulvin (GRIS){0.015–8 μg/mL}, fluconazole (FLC){0.125–64 μg/mL}, terbinafine (TRB){0.06–32 μg/mL}, posaconazole (POS) {0.015 to 8 μg/mL}, LULI {0.0035 to 2 μg/mL}. The MICs of azoles, GRIS, and amorolfine were documented at the concentration showing prominent inhibition of growth (approximately 80%) compared to that in growth control wells. For TRB and LULI, a 100% growth inhibition was documented.[2,8]
The growth in each well was compared with growth control with the aid of a reading mirror. MIC50 was calculated by taking the drug concentration, where 50% of isolates were inhibited. Similarly, MIC90 was noted with drug concentration where 90% of the isolates were inhibited.[8] Reference strain used Candida parapsilosis ATCC® 22019.
Clinical significance: There are no breakpoints established for antifungal agents against dermatophytes. The interpretive criteria were assessed according to breakpoints established in CLSI M38-A2 reference documents and other studies.[8,9]
Statistical analysis was performed by Chi-square tests using the SPSS-20 software to find significant differences between variables. Comparison of continuous variables between the two groups was conducted using the independent t-test and more than two group comparison was conducted using one-way analysis of variance (ANOVA). A P value of < 0.05 was considered significant.
Results
Out of 236 clinically diagnosed dermatophytic patients, 113 (47.9%) were males and 123 (52.1%) were females. The age range of patients was 1–85 years. Also, 26.6% were positive for both KOH and culture and 36% (85) were positive for fungal hyphae by microscopy with KOH mount showing the abundance of hyaline, long, smooth, septate, branching hyphae whereas 26.6% (63) were positive for both KOH and culture. The culture was positive in 48% of the cases for dermatophytes, non-dermatophytic molds and yeasts and in 16% (38) cases for dermatophytes only. Non-dermatophytic molds were considered pathogenic after isolates cultured from repeated samples in our lab. Trichophyton rubrum (28.95%) was the most predominant dermatophyte species isolated [Figure 1]. The gross and microscopic morphology shown [Figure 2]. The maximum samples collected from patients were nail clippings and scrapings [Table 1a].
Figure 1.
Dermatophytes distribution
Figure 2.
Gross and microscopic morphology of dermatophytes. (a) Colonies of T. rubrum appear fluffy and reverse is deep red color on SDA tubes; (b) LPCB mount of T. rubrum showing tear drop microconidia and pencil like macroconidia (Magnification 40X); (c) Growth of M. canis appearing whitish, coarsely fluffy with yellow pigment on SDA tubes; (d) LPCB mount of M. canis showed spindle shaped, rough, and thick walled macroconidia with taper to knob-like ends. (e) Colonies of T. mentagrophytes appearing powdery, white and downy on SDA (Sabourad’s Dextrose agar) tube; (f) LPCB mount showing septate hyphae, coiled spiral hyphae and cluster of conidiophores of T. mentagrophytes; (g) Colonies of E. floccosum producing greenish-brown or khaki-colored colonies, with a suede-like surface on SDA (Sabourad’s Dextrose agar) tube; (h) LPCB mount showing smooth, thin-walled macroconidia occurring in clusters on the hyphal threads of E. floccosum
Table 1a.
Distribution of dermatophytes isolated from different samples
| Clinical | T. ment | T. rub | T. verr | T. tons | T. viol | M. canis | M. gyp | E. floc |
|---|---|---|---|---|---|---|---|---|
| Skin | 4 (40%) | 3 (27%) | 1 (50%) | 3 (30%) | 1 (50%) | 1 (100%) | 0 | 0 |
| Hair | 1 (10%) | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Nail | 5 (50%) | 8 (72.7%) | 1 (50%) | 7 (70%) | 1 (50%) | 0 | 1 (100%) | 1 (100%) |
| Total | 10 | 11 | 2 | 10 | 2 | 1 | 1 | 1 |
The most common causative agents for Tinea corporis, Tinea unguium, and Tinea capitis were Trichophyton tonsurans, T. rubrum and T. mentagrophytes, respectively [Table 1b]. In this study, the MIC range, MIC90 and geometric mean values of eight antifungals for dermatophytosis were compared with many studies conducted over recent years to assess the current susceptibility pattern. Table 2 shows T. rubrum had the highest geometric mean MIC for FLC (7.511 μg/mL) followed by GRIS (1.554 μg/mL) and the lowest for LULI (0.004 μg/mL). For the isolates of T. mentagrophytes and T. tonsurans, GM MIC was the highest for FLC (22.6 μg/mL and 27.857 μg/mL, respectively) and the lowest for LULI (0.004 μg/mL and 0.009 μg/mL, respectively). The two isolates of T. verrucosum had high MIC values for FLC and TRB (2–8 μg/mL and 8–16 μg/mL, respectively). The two isolates of T. violaceum had high MIC values for FLC and GRIS (16–32 μg/mL and 4–8 μg/mL, respectively). Overall, MIC90 for all isolates was the highest for FLC and the lowest for LULI. Table 3 shows that among the 11 isolates of T. rubrum, 36.3% of isolates had MIC values ≥4 μg/mL and so were resistant for GRIS and FLC. Among the 10 isolates of T. tonsurans isolates, 50% were resistant to GRIS and 80% to FLC. Among the 10 isolates of T. mentagrophytes, 60% were resistant to GRIS and 50% to FLC. All isolates of T. violaceum were resistant to GRIS and FLC. All isolates of T. verrucosum were resistant to TRB. Single isolates of Microsporum spp. and E. floccosum were resistant to GRIS and FLC.
Table 1b.
Distribution of dermatophytes isolated from different clinical presentations
| Clinical | T. ment | T. rub | T. verr | T. tons | T. viol | M. canis | M. gyp | E. floc |
|---|---|---|---|---|---|---|---|---|
| T. corporis | 2 | 2 | 0 | 3 | 1 | 1 | 0 | 0 |
| T. unguium | 3 | 7 | 1 | 4 | 1 | 0 | 1 | 0 |
| T. cruris | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
| T. capitis | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| T. barbae | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| T. mannum | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| T. pedis | 2 | 1 | 0 | 3 | 0 | 0 | 0 | 1 |
| Total | 10 | 11 | 2 | 10 | 2 | 1 | 1 | 1 |
Table 2.
MIC (MIC50, MIC 90, MIC range, and geometric mean) of antifungal drugs against dermatophytes species
| Organism | Drugs MIC* | POS | LULI | |||||
|---|---|---|---|---|---|---|---|---|
|
| ||||||||
| TRB | KTC | GRIS | FLC | ITC | VRC | |||
| T. rubrum (n=11) | ||||||||
| GM | 0.220 | 0.115 | 1.554 | 7.511 | 0.140 | 0.095 | 0.089 | 0.004 |
| MIC50 | 0.25 | 0.125 | 2 | 16 | 0.125 | 0.06 | 0.125 | 0.003 |
| MIC90 | 0.5 | 0.5 | 8 | 32 | 0.25 | 0.25 | 0.125 | 0.015 |
| Range | 0.125-0.5 | 0.03-0.5 | 0.25-8 | 0.5-64 | 0.03-0.25 | 0.03-0.5 | 0.03-0.5 | 0.003-0.015 |
| T. tonsurans (n=10) | ||||||||
| GM | 1.231 | 0.304 | 2.462 | 27.857 | 0.25 | 0.615 | 0.107 | 0.009 |
| MIC50 | 1 | 0.25 | 2 | 32 | 0.25 | 0.5 | 0.125 | 0.007 |
| MIC90 | 8 | 2 | 8 | 64 | 0.5 | 2 | 0.25 | 0.06 |
| Range | 0.25-16 | 0.03-2 | 0.5-8 | 2-64 | 0.125-0.5 | 0.125-16 | 0.06-0.25 | 0.007-0.06 |
| T. mentagrophytes (n=10) | ||||||||
| GM | 0.267 | 0.197 | 1.587 | 22.627 | 0.123 | 0.268 | 0.089 | 0.004 |
| MIC50 | 0.25 | 0.25 | 4 | 16 | 0.125 | 0.25 | 0.06 | 0.003 |
| MIC90 | 0.5 | 0.5 | 8 | 64 | 0.25 | 0.5 | 0.25 | 0.007 |
| Range | 0.06-2 | 0.03-1 | 0.125-8 | 8-64 | 0.03-0.5 | 0.06-1 | 0.03-0.5 | 0.003-0.015 |
| T. violaceum (n=2) | ||||||||
| Range | 0.5-1 | 0.5-1 | 4-8 | 16-32 | 0.03-0.06 | 0.25-0.5 | 0.06-0.5 | 0.003-0.007 |
| T. verrucosum (n=2) | ||||||||
| Range | 8-16 | 0.25-0.5 | 1-2 | 2-8 | 0.25-0.5 | 0.5-1 | 0.25-0.5 | 0.007-0.06 |
| M. canis (n=1) | ||||||||
| MIC | 0.5 | 0.125 | 4 | 32 | 0.25 | 0.25 | 0.06 | 0.003 |
| M. gypseum (n=1) | ||||||||
| MIC | 8 | 1 | 8 | 64 | 1 | 1 | 0.25 | 0.003 |
| E. floccosum (n=1) | ||||||||
| MIC | 2 | 1 | 8 | 32 | 0.5 | 0.25 | 0.125 | 0.007 |
VRC=voriconazole, FLC=fluconazole, KTC=ketoconazole, ITC=itraconazole, POS=posaconazole, LULI=luliconazole, TRB=terbinafine, GRIS=griseofulvin
Table 3.
MIC values of antifungal drugs for dermatophytes isolates
| Specimen | Drugs | 0.003 | 0.007 | 0.015 | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| T. rub (11) | TRB | 5 (45) | 3 | 3 | 0 | |||||||||||
| KTC | 2 (18) | 2 (18) | 4 (36) | 1 (9) | 2 | |||||||||||
| GRIS | 2 | 2 | 3 | 2 | 2 | |||||||||||
| FLC | 1 | 1 | 2 | 1 | 2 | 3 | 1 | |||||||||
| ITC | 1 | 1 | 4 | 5 | ||||||||||||
| VRC | 3 | 3 | 1 | 3 | 1 | |||||||||||
| POS | 2 | 3 | 5 (45) | 1 | ||||||||||||
| LULI | 7 (63) | 2 | 2 | |||||||||||||
| T. tons (10) | TRB | 2 | 2 | 2 | 2 | 1 | 1 | 0 | ||||||||
| KTC | 2 | 1 | 2 | 1 | 2 | 2 | ||||||||||
| GRIS | 2 | 3 | 3 | 2 | ||||||||||||
| FLC | 1 | 1 | 3 | 5 | ||||||||||||
| ITC | 3 | 4 | 3 | |||||||||||||
| VRC | 1 | 2 | 4 | 2 | 1 | |||||||||||
| POS | 4 | 4 | 2 | |||||||||||||
| LULI | 3 | 4 | 1 | 2 | ||||||||||||
| T. ment (10) | TRB | 2 | 5 (50) | 2 | 1 | 0 | ||||||||||
| KTC | 1 | 3 | 4 | 1 | 1 | |||||||||||
| GRIS | 1 | 2 | 1 | 2 | 4 | |||||||||||
| FLC | 1 | 1 | 3 | 1 | 4 | |||||||||||
| ITC | 2 | 4 | 3 | 1 | ||||||||||||
| VRC | 1 | 2 | 2 | 3 | 2 | |||||||||||
| POS | 2 | 3 | 3 | 1 | 1 | |||||||||||
| LULI | 5 (50) | 4 (40) | 1 (10) | |||||||||||||
| T. viol (2) | TRB | 1 | 1 | 0 | ||||||||||||
| KTC | 1 | 1 | 0 | |||||||||||||
| GRIS | 1 | 1 | ||||||||||||||
| FLC | 1 | 1 | ||||||||||||||
| ITC | 1 | 1 | ||||||||||||||
| VRC | 1 | 1 | ||||||||||||||
| POS | 1 | 1 | ||||||||||||||
| LULI | 1 (50) | 1 (50) | ||||||||||||||
| T. verr (2) | TRB | 1 | 1 | |||||||||||||
| KTC | 1 | 1 | ||||||||||||||
| GRIS | 1 | 1 | 0 | |||||||||||||
| FLC | 1 | 1 | ||||||||||||||
| ITC | 1 | 1 | ||||||||||||||
| VRC | 1 | 1 | ||||||||||||||
| POS | 1 | 1 | ||||||||||||||
| LULI | 1 | 1 | ||||||||||||||
| M. canis (1) | TRB | 1 | 0 | |||||||||||||
| KTC | 1 | |||||||||||||||
| GRIS | 1 | |||||||||||||||
| FLC | 1 | |||||||||||||||
| ITC | 1 | |||||||||||||||
| VRC | 1 | |||||||||||||||
| POS | 1 | |||||||||||||||
| LULI | 1 | |||||||||||||||
| M. gyps (1) | TRB | 1 | ||||||||||||||
| KTC | 1 | |||||||||||||||
| GRIS | 1 | |||||||||||||||
| FLC | 1 | |||||||||||||||
| ITC | 1 | |||||||||||||||
| VRC | 1 | |||||||||||||||
| POS | 1 | |||||||||||||||
| LULI | 1 | |||||||||||||||
| E. flocc (1) | TRB | 1 | ||||||||||||||
| KTC | 1 | |||||||||||||||
| GRIS | 1 | |||||||||||||||
| FLC | 1 | |||||||||||||||
| ITC | 1 | |||||||||||||||
| VRC | 1 | |||||||||||||||
| POS | 1 | |||||||||||||||
| LULI | 1 |
T. rub=Trichophyton rubrum, T. ment=Trichophyton mentagrophytes, T. viol=Trichophyton violaceum, T. verr=Trichophyton verrucosum, T. tons=Trichophyton tonsurans, M. canis=Microsporum canis, M. gyps=Microsporum gypseum, E. flocc=Epidermophyton floccosum
As per the study of Pathania et al.,[9] we have considered a MIC ≥ 4 μg/mL for an isolate to be labelled as resistant to TRB, ITC, GRIS and MIC ≥ 32 μg/mL to be resistant to FLC.
Discussion
The prevalence of dermatophytosis in the present study was found to be 16%, which falls within the current global range.[1] Trichophyton rubrum was the predominant isolate in our study, which is in accordance with the results of Vineetha and Saxena et al.[10,11] T. rubrum still remains the commonest dermatophyte in most regions of the world even though the epidemiology is changing. Jha et al.[12] reported T. rubrum as the most common etiological agent of Tinea unguium/onychomycosis due to poor personal hygiene and use of detergent in females, same as our study. Kaur et al.[13] found a higher incidence of T. tonsurans in cases of Tinea corporis from sharing of fomites as also observed in this study (7.8%). Gupta and Budhiraja et al. have reported T. mentagrophytes as the most common pathogen due to newer genotypes with fungal resistance secondary to the abuse of irrational fixed-dose combination creams.[14,15]
The recent epidemic-like scenario in India and emerging case reports of recalcitrant tinea have highlighted the importance of AFST for dermatophytes. Different techniques, such as disc diffusion, agar dilution, macro and micro broth dilution methods, are available.[6] The microdilution assay for dermatophytes is convenient and reproducible although the preparation of conidia inoculum is sometimes a challenge with dermatophytes. Clinical breakpoints (CBP) have not yet been established by CLSI for mould testing.[8]
LULI, POS, VRC, ITC and KTC showed good activity against all isolates of T. rubrum, T. tonsurans and T. mentagrophytes. LULI was found to have the lowest MIC values against all dermatophytes, indicating good in vitro activity but is not commonly used in clinical practice yet.[14] Tahiliani et al. found that LULI demonstrated the lowest mean MIC values across different regions, particularly Delhi and Kolkata.[16,17] LULI has shown better efficacy against filamentous fungi; so, currently, it is preferred as a topical agent.
In our study, POS has a very low MIC range (0.03–0.5 μg/mL) for all isolates, similar to the results of Badali et al.[17] In comparison to other routinely used azoles, POS and LULI were found to be better drugs as they have better in vitro susceptibility.
MIC range for all isolates of ITC in our study (0.03–1 μg/mL) was found to conform with the findings of Kumar et al. (0.03–0.125 μg/mL), Budhiraja et al.(0.03–0.125 μg/mL) for T. rubrum and T. mentagrophyte isolates.[15,18] Similar to Deng et al.,[19] our study reported lower MIC of VRC indicating higher susceptibility as it is rarely used. Other studies reported MIC ranges of 0.015–16 μg/mL for T. rubrum indicating that some strains showed resistance to ITC.[19] Badali et al.[17] have also shown higher MIC90 of ITC as compared to our study. The novel squalene epoxidase substitution Ala448Thr was associated with higher MICs of ITR and VRC causing triazole insensitivity in recent studies.[20]
The MIC90 of VRC was the highest for T. tonsurans (4 μg/mL) and the lowest for M. canis and E. floccosum at 0.25 μg/mL, which corresponds with the findings of Shaw et al.[21] In the study of Sardana et al.,[7] VRC had the lowest MIC values among all the drugs tested. The reason could be that being a relatively more expensive drug it is less abused. Also, the serum levels vary widely due to varying metabolism, increasing the risk of toxicity or therapeutic failure and it achieves very poor skin levels and may not have in vivo utility. As a result, it is less used in clinical practice.
In the present study, the GM MIC of TRB was 0.0115 μg/mL for T. rubrum and 0.197 μg/mL for T. mentagrophytes, indicating that these drugs had good activity against these species. All isolates were sensitive except T. verrucosum and M. gypseum, which were resistant to TRB. Our study observed a higher MIC range (0.06–16 μg/mL) probably due to the resistance of two isolates to TRB, similar to the findings of Budhiraja et al.[15] High TRB resistance was attributed to frequent usage in common practice for decades and squalene epoxidase (SQLE) point mutations.[22] The activity of TRB was found to be species-dependent. Low GM MIC for TRB was observed in all dermatophytes such as in Gupta et al’s study.[14] Carrillo-Muñoz et al.[23] have reported excellent TRB activity against isolates of T. rubrum (GM-0.26) and T. mentagrophytes and resistance to M. gypseum, which correlates well with our findings.
A higher MIC range (0.125–8 μg/mL) for all isolates was observed in our study for GRIS, in accordance with other studies.[14,24] It has been the first-line drug for a long time before azoles and this has led to high MIC values observed recently. The treatment failure with GRIS resulted in allylamines becoming the preferred choice.[7] GRIS showed a high MIC90 in this study for all isolates of T. rubrum, T. tonsurans and T. mentagrophytes. Therefore, combination therapy is promoted for higher cure rates, for example, itraconazole + griseofulvin (93.1%) and terbinafine + griseofulvin (78.5%).[25] Kumar et al. and Das showed relatively good sensitivity to GRIS against strains of T. verrucosum and T. rubrum similar to our study.[18,26]
Our study reported poor susceptibility to FLC with very high MIC values similar to other studies.[25,27] Also, 52.6% of the isolates were resistant to FLC (CBP MIC ≥ 32 μg/mL). Over-the-counter preparations easily available in India and irrational usage by quacks probably contribute to high resistance against FLC.[19] ABC transporter gene is a major factor in the development of resistance to azoles in T. rubrum.[2] Triazole resistance especially FLC is high (35.4%) in the Indian isolates, a big reason for treatment failure.[14] Recent studies have found nano-liposomal fluconazole and nano-liposomal terbinafine better than the conventional forms for dermatophytosis treatment, which can be used in the future.[28]
This study displayed excellent activities against dermatophyte species by all newer azoles except FLC.
It must be noted that the correlation between in vitro results and clinical outcomes of cases of dermatophytosis is still to be established. Neither MIC50 nor MIC90 can categorize the isolates as resistant or sensitive as they only indicate the MIC of 50% or 90% of the tested isolates. This information is used to map the susceptibility pattern of the circulating strains in a given setting (institute, region, or country, etc.). Keeping in mind the changing epidemiology and increasing resistance in dermatophytes, more studies based on species distribution and AFST are needed to understand and contain the growing menace of dermatophytosis.
Limitations
The sample size was small and the study did not include an in vivo correlation of responsiveness to drugs due to the short duration of the study period. Genomic studies on isolated species would have given us an insight into possible mutations. However, this was not possible in view of cost and infrastructure constraints.
Conclusion
The current study attempted to ascertain the most prevalent dermatophytes causing a major burden on our community and determine their AFST. A comparative analysis of MIC values of newer antifungals such as LULI, POS, VRC with commonly used drugs was conducted. Azoles (ITC, VRC, KTC, POS and LULI) had lower MIC90, indicating that they had good in vitro activity against dermatophytes and would be effective in clinically diagnosed cases of Tinea or dermatophytosis. In contrast, the commonly used antifungals such as FLC, GRIS and TRB showed high MIC values. Although the sample size was small, these data provide valuable insights into potential alternative treatment options against relapse/recalcitrant/failure cases and can serve as a pilot for studies on a larger scale to provide data that would contribute to improving treatment protocols and patients’ quality of life.
Declaration of patient consent
Written informed consent was obtained from all individual participants included in the study.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
The authors would like to thank the staff of Mycology Laboratory, LHMC, for their help in the procedures required for the diagnosis.
Funding Statement
Nil.
References
- 1.Lakshmanan A, Ganeshkumar P, Mohan SR, Hemamalini M, Madhavan R. Epidemiological and clinical pattern of dermatomycoses in rural India. Indian J Med Microbiol. 2015;33((Suppl)):134–6. doi: 10.4103/0255-0857.150922. [DOI] [PubMed] [Google Scholar]
- 2.Rudramurthy S, Shaw D. Overview and update on the laboratory diagnosis of dermatophytosis. Clin Dermatol Rev. 2017;1:3. [Google Scholar]
- 3.Grumbt M, Monod M, Staib P. Genetic advances in dermatophytes. FEMS Microbiol Lett. 2011;320:79–86. doi: 10.1111/j.1574-6968.2011.02276.x. [DOI] [PubMed] [Google Scholar]
- 4.Maurya VK, Kachhwaha D, Bora A, Khatri PK, Rathore L. Determination of antifungal minimum inhibitory concentration and its clinical correlation among treatment failure cases of dermatophytosis. J Family Med Prim Care. 2019;8:2577–81. doi: 10.4103/jfmpc.jfmpc_483_19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bhatia VK, Sharma PC. Determination of minimum inhibitory concentrations of itraconazole, terbinafine and ketoconazole against dermatophyte species by broth microdilution method. Indian J Med Microbiol. 2015;33:533–7. doi: 10.4103/0255-0857.167341. [DOI] [PubMed] [Google Scholar]
- 6.Dogra S, Shaw D, Rudramurthy SM. Antifungal drug susceptibility testing of dermatophytes: Laboratory findings to clinical implications. Indian Dermatol Online J. 2019;10:225–33. doi: 10.4103/idoj.IDOJ_146_19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Sardana K, Kaur R, Arora P, Goyal R, Ghunawat S. Is antifungal resistance a cause for treatment failure in dermatophytosis: A study focused on tinea corporis and cruris from a tertiary centre? Indian Dermatol Online J. 2018;9:90–5. doi: 10.4103/idoj.IDOJ_137_17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Clinical Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi;Approved Standard, 3rd ed. CLSI document M38 2018 Wayne, PA, USA Clinical and Laboratory Standards Institute. 2018 [Google Scholar]
- 9.Pathania S, Rudramurthy SM, Narang T, Saikia UN, Dogra S. A prospective study of the epidemiological and clinical patterns of recurrent dermatophytosis at a tertiary care hospital in India. Indian J Dermatol Venereol Leprol. 2018;84:678–84. doi: 10.4103/ijdvl.IJDVL_645_17. [DOI] [PubMed] [Google Scholar]
- 10.Vineetha M, Sheeja S, Celine MI, Sadeep MS, Palackal S, Shanimole PE, et al. Profile of dermatophytosis in a tertiary care center. Indian J Dermatol. 2018;63:490–5. doi: 10.4103/ijd.IJD_177_18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Saxena G, Sadawarte K, Songara P, Mehta A. Clinico-mycological profile of dermatophytoses at a tertiary care teaching hospital of central India. SVU-Int J Med Sci. 2022;5:216–27. [Google Scholar]
- 12.Jha B, Sharma M, Gc S, Sapkota J. Onychomycosis among clinically suspected cases attending the dermatology out-patient department of a tertiary care centre: A descriptive cross-sectional study. JNMA J Nepal Med Assoc. 2021;59:450–3. doi: 10.31729/jnma.6277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kaur I, Chaudhary A, Singh H. Clinico-microbiological aspects of tinea corporis in North India: Emergence of Trichophyton tonsurans. Int J Res Dermatol. 2019;5:144. [Google Scholar]
- 14.Gupta AK, Adam P, Dlova N, Lynde CW, Hofstader S, Morar N, et al. Therapeutic options for the treatment of tinea capitis caused by Trichophyton species: Griseofulvin versus the new oral antifungal agents, terbinafine, itraconazole, and fluconazole. Pediatr Dermatol. 2001;18:433–8. doi: 10.1046/j.1525-1470.2001.01978.x. [DOI] [PubMed] [Google Scholar]
- 15.Budhiraja RK, Sharma S, Sharma S, Kaur J, Bassi R. Antifungal susceptibility pattern of dermatomycosis in a tertiary care hospital of North India. Int J Res Dermatol. 2018;4:240. [Google Scholar]
- 16.Tahiliani S, Saraswat A, Lahiri AK, Shah A, Hawelia D, Shah GK, et al. Etiological prevalence and antifungal sensitivity patterns of dermatophytosis in India-A multicentric study. Indian J Dermatol Venereol Leprol. 2021;87:800–6. doi: 10.25259/IJDVL_1025_19. [DOI] [PubMed] [Google Scholar]
- 17.Badali H, Mohammadi R, Mashedi O, de Hoog GS, Meis JF. In vitro susceptibility patterns of clinically important trichophyton and epidermophyton species against nine antifungal drugs. Mycoses. 2015;58:303–7. doi: 10.1111/myc.12315. [DOI] [PubMed] [Google Scholar]
- 18.Kumar MB, Gulati N, Chander J, Singla N, Bhalla M, Nayyar C, et al. Species Distribution and Antifungal Susceptibility Profile of Dermatophytes from a Tertiary Care Centre in North India. J Lab Physicians. 2022;14:449–55. doi: 10.1055/s-0042-1748826. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Deng S, Zhang C, Seyedmousavi S, Zhu S, Tan X, Wen Y, et al. Comparison of the in vitro activities of newer triazoles and established antifungal agents against Trichophyton rubrum. Antimicrob Agents Chemother. 2015;59:4312–4. doi: 10.1128/AAC.00244-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Ebert A, Monod M, Salamin K, Burmester A, Uhrlaß S, Wiegand C, et al. Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicentre study. Mycoses. 2020;63:717–28. doi: 10.1111/myc.13091. [DOI] [PubMed] [Google Scholar]
- 21.Shaw D, Singh S, Dogra S, Jayaraman J, Bhat R, Panda S, et al. MIC and upper limit of wild-type distribution for 13 antifungal agents against a trichophyton mentagrophytes-trichophyton interdigitale complex of Indian origin. Antimicrob Agents Chemother. 2020;64:e01964–19. doi: 10.1128/AAC.01964-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Khurana A, Masih A, Chowdhary A, Sardana K, Borker S, Gupta A, et al. Correlation of in vitro susceptibility based on MICs and squalene epoxidase mutations with clinical response to terbinafine in patients with tinea corporis/cruris. Antimicrob Agents Chemother. 2018;62:e01038–18. doi: 10.1128/AAC.01038-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Carrillo-Muñoz AJ, Giusiano G, Cárdenes D, Hernández-Molina JM, Eraso E, Quindós G, et al. Terbinafine susceptibility patterns for onychomycosis-causative dermatophytes and Scopulariopsis brevicaulis. Int J Antimicrob Agents. 2008;31:540–3. doi: 10.1016/j.ijantimicag.2008.01.023. [DOI] [PubMed] [Google Scholar]
- 24.Galuppi R, Gambarara A, Bonoli C, Ostanello F, Tampieri MP. Antimycotic effectiveness against dermatophytes: Comparison of two in vitro tests. Vet Res Commun. 2010;34((Suppl 1)):S57–61. doi: 10.1007/s11259-010-9386-1. [DOI] [PubMed] [Google Scholar]
- 25.Vanapalli S, Turpati NR, Gopal KVT, Krishnam Raju PV, Devi BG. A clinico-mycological, antifungal drug sensitivity and therapeutic study of extensive dermatophytosis in coastal Andhra Pradesh. Indian Dermatol Online J. 2022;13:747–53. doi: 10.4103/idoj.idoj_143_22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Das S, De A, Saha R, Sharma N, Khemka M, Singh S, et al. The Current Indian Epidemic of Dermatophytosis: A study on causative agents and sensitivity patterns. Indian J Dermatol. 2020;65:118–22. doi: 10.4103/ijd.IJD_203_19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Fernández-Torres B, Carrillo AJ, Martín E, Del Palacio A, Moore MK, Valverde A, et al. In vitro activities of 10 antifungal drugs against 508 dermatophyte strains. Antimicrob Agents Chemother. 2001;45:2524–8. doi: 10.1128/AAC.45.9.2524-2528.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Musavi Bafrui N, Hashemi Hazaveh SJ, Bayat M. In-vitro activity of nano fluconazole and conventional fluconazole against clinically important dermatophytes. Iran J Public Health. 2020;49:1970–6. doi: 10.18502/ijph.v49i10.4701. [DOI] [PMC free article] [PubMed] [Google Scholar]