Abstract
Background:
The widespread occurrence of chronic and recurrent dermatophytosis has significantly affected the quality of life for patients in India and beyond. Identifying the causative dermatophytes and understanding their antifungal susceptibility can aid clinicians in tailoring effective antifungal therapies.
Materials and Methods:
Patients with chronic and recurrent dermatophytosis were enrolled, and conventional fungal cultures were conducted on skin scrapings. Identified isolates underwent antifungal susceptibility testing using the Clinical and Laboratory Standards Institute broth microdilution method (CLSI M38-A2) for common systemic antifungals, determining the minimum inhibitory concentration (MIC) range and calculating MIC 50 and MIC 90.
Results:
Sixty samples were tested. Tinea corporis was the most common presentation (66.6%). Trichophyton mentagrophyte species complex was the prevalent species (45, 75%), followed by Trichophyton rubrum (7, 11.7%). In Trichophyton mentagrophytes species complex, MIC range was 8-64 μg/mL for fluconazole, 0.06-0.25 μg/mL for terbinafine, and 0.125-0.5 μg/mL for griseofulvin. For Trichophyton rubrum, the MIC range was 8-64 μg/mL for fluconazole, 0.06-0.25 μg/mL for terbinafine, and 0.125-0.5 μg/mL for griseofulvin. For all species, itraconazole MIC was ≤0.125 μg/mL. Hence, itraconazole and terbinafine had the best MIC range against tested isolates in our study.
Limitations:
Absence of genotyping of isolate and not compared the results with studies where sequence-based identification to species level was done.
Conclusion:
In vitro, resistance to itraconazole for any of the four isolated agents was not seen. Terbinafine resistance appears to be an uncommon occurrence in South India. In vitro susceptibility tests shall be regularly done to design the epidemiological cutoff values.
Keywords: Antifungal agents, antifungal resistance, azole, dermatophytes, terbinafine
Introduction
Dermatophyte infections are common and worldwide, affecting 20-25% of global population, especially in tropical and subtropical regions. Various genera, including Trichophyton, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, Microsporum, and Arthroderma, have been identified by molecular methods as the cause of infection.[1] In India, there is a notable rise in chronic and recurrent cases, often unresponsive to standard antifungal treatments.[1] These cases comprise 25-30% of all dermatophytosis cases and are challenging to manage, necessitating new guidelines.[2] Recalcitrant dermatophytosis encompasses relapse, recurrence, reinfection, persistence, and potential resistance to treatment.[3]
The reasons for treatment failure in dermatophytosis include inadequate treatment duration, difficulties in eliminating predisposing factors, reinfection, and emerging antifungal resistance.[3,4] Accurate identification of the organism to the species level, along with in vitro antifungal susceptibility testing (AFST), is crucial for effective therapy.[1] However, routine testing is limited due to the high load of cases and time constraints in laboratories. Therefore, there is a need for antifungal susceptibility studies across the country to understand prevailing trends. Traditional methods for dermatophyte identification are laborious, and molecular techniques are now considered necessary. In this study, we have employed clinical and conventional mycological methods for species identification, followed by in vitro antifungal susceptibility testing. We also determined the minimum inhibitory concentrations (MICs) of four commonly used antifungals, including MIC 50 and MIC 90 values. These require significant scientific expertise for precise identification of the organism.[5] Previously, species biotypes went unidentified as a result of the limited availability of molecular techniques. Traditional taxonomical classification is deemed inadequate for this fungal group; a new molecular-based approach is explored.[6]
Materials and Methods
The study, which was authorized by the institution’s ethics committee, acquired signed agreement from all patients who took part. Patients aged >18 years with chronic and recurrent dermatophytosis were screened using direct microscopy (potassium hydroxide mount). Patients with chronic and recurrent dermatophytosis were recruited. Chronic dermatophytosis was defined as glabrous tinea persisting for six months or longer, with or without treatment. Glabrous tinea reoccurring four weeks after stopping antifungal treatment following clinical cure was defined as a recurrent type.[3]
Skin scrapings were collected from the lesion and transported to the laboratory. They were subjected to mycological culture on Sabouraud dextrose agar (SDA) with chloramphenicol and cycloheximide (SCC). AFST was done using the standard broth microdilution method using the Clinical and Laboratory Standards Institute (CLSI)-M38-A2 guidelines document.[7] Trichophyton mentagrophytes (ATCC 4439) was employed as the quality control strain. All antifungal drugs were acquired as reagent-grade powders from Sigma Aldrich, St. Louis, Missouri, USA. RPMI-1640 medium (Roswell Park Memorial Institute Medium) containing L-glutamine and without bicarbonate (pH 7.0, corrected with 0.165 M of morpholinepropanesulfonic acid) was used.[8] The drugs were prepared at final concentrations using serial two-fold dilutions. Inoculum concentration ranged from 1 × 103 to 3 × 103 colony forming unit/mL. Drug stocks were diluted two-fold in RPMI-1640 in 96-well plates; 100 μL of inoculum suspension containing conidia and RPMI media was added to each well. Plates were then incubated at 35°C for up to 7 days without shaking. MIC 50 and 90 were determined by identifying the drug concentrations inhibiting 50% and 90% of the isolate, respectively.[9] The minimum inhibitory concentration for antifungal drugs was the lowest concentration causing significant growth inhibition (approximately 80% or more compared to the control well).[10] These concentrations were calculated in Microsoft Excel (version 2007) and analyzed using SPSS (version 22.0).
Results
Sixty dermatophyte isolates were randomly selected from clinical specimens of patients with chronic and recurrent dermatophytosis. Of these, 36 (60%) were female and 24 (40%) were male, identified through positive direct microscopy. The majority of patients were adults (25-45 years, 44, 73.3%), followed by adolescents (18-25 years, 16, 26.7%). Tinea corporis was the most prevalent clinical condition (40, 66.6%), followed by tinea cruris (20, 33.3%) [Figure 1]. The most common isolate was Trichophyton mentagrophytes species complex (T. mentagrophytes) (45, 75%), followed by Trichophyton rubrum (T. rubrum) (7, 11.7%), Nannizzia gypsea (N. gypsea) (6, 10%), and Microsporum canis (M. canis) (2, 3.3%) [Figures 2 and 3]. Itraconazole exhibited sensitivity in 60 isolates, with consistently recorded MIC values ≤0.125 μg/mL, thus confirming its efficacy in combating the tested organisms. The MICs for T. mentagrophytes species complex ranged from 8 to 64 μg/mL for fluconazole, 0.06 to 0.25 μg/mL for terbinafine, and 0.125 to 0.5 μg/mL for griseofulvin. In T. rubrum, 8-64 μg/mL for fluconazole, 0.06-0.25 μg/mL for terbinafine, and griseofulvin ranged from 0.125 to 0.5 μg/mL in our study. In N. gypsea and M. canis, fluconazole ranged from 2 to 64 μg/mL and 4 to 64 μg/mL, respectively.
Figure 1.
(a and b) Multiple confluent and diffuse annular plaques of tinea corporis manifesting as chronic dermatophytosis
Figure 2.
Growth of dermatophytes on Sabouraud dextrose agar. Trichophyton mentagrophytes colonies; flat, creamy, and granular surface (a) with reverse yellow-brown pigmentation (b), Trichophyton rubrum colonies; flat, creamy, and downy surface (c) with reverse red pigmentation (d), Nannizzia gypsea colonies; flat, creamy, and granular (e) with reverse yellow-brown pigmentation (f), Microsporum canis; a white to a yellowish colony with a coarsely fluffy, velvety/powdery texture (g) having a bright golden yellow to brownish-yellow on the reverse (h)
Figure 3.
Tease mount microscopy using lactophenol cotton blue stain, (40×). (a) T. mentagrophytes showing numerous club-shaped microconidia along with the spiral hyphae. (b) T. rubrum showing slender clavate to pyriform, and tear-shaped microconidia (c). N. gypsea showing rough-walled, blunt, club-shaped, multicelled macroconidia. (d) M. canis showing spindle-shaped, multicelled, verrucose, thick-walled macroconidia with terminal knob
In T. mentagrophytes species complex isolates, the MIC values for fluconazole were as follows: 8 μg/mL in 25 isolates (55.5%), 16 μg/mL in 16 isolates (35.6%), and 64 μg/mL in 4 isolates (8.9%). Among T. rubrum isolates, 2 isolates (28.6%) displayed MIC values of 64 μg/mL for fluconazole, while the remaining five isolates (71.4%) showed 8 μg/mL. Notably, both N. gypsea and M. canis exhibited a minimum MIC of 2 and 4 μg/mL, respectively.
Among T. mentagrophytes species complex isolates, terbinafine exhibited MIC values of 0.25 μg/mL in 4 isolates (8.9%), 0.125 μg/mL in 21 isolates (46.7%), and 0.06 μg/mL in 20 isolates (44.4%). Regarding T. rubrum, MIC values were observed at 0.25 µg/mL in 2 isolates (28.6%), 0.125 µg/mL in 2 isolates (28.6%), and 0.06 µg/mL in 2 isolates (28.6%), and one isolate was not assessed for terbinafine due to technical error. The MIC values signifying sensitivity to the drug can be considered below 0.06 μg/ml for both N. gypsea and M. canis, however, we have not tested for values below o.o6 μg. Griseofulvin showed MIC values of 0.5 μg/mL for 12 (26.7%) isolates of T. mentagrophytes species complex, 0.25 μg/mL for 24 (53.3%) isolates, and 0.125 μg/mL for 9 (20%) isolates. For T. rubrum, MIC values were 0.5 μg/mL in 2 (28.5%) isolates, 0.25 μg/mL in 4 (57.1%) isolates, and 0.125 μg/mL in one (14.3%) isolate. However, N. gypsea and M. canis were not tested for griseofulvin Table 1. The MIC 50 and MIC 90 for T. mentagrophytes species complex and T. rubrum are shown in Table 2. It is noteworthy that itraconazole was found to have MIC values of below ≤0.125μg/mL for all tested isolates, hence, MIC 50 and MIC 90 was not calculated.
Table 1.
The MIC for four antifungal agents tested against 60 dermatophyte isolates by the CLSI broth microdilution methods (mg/L)
| No. of isolates | Antifungal | MIC Distribution (µg/mL) (No. isolates with %) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||||
| 64 | 32 | 16 | 8 | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 | 0.06 | ||
| Tm (45) | GRI | x | x | 12 (26.7) | 24 (53.3) | 9 (20) | ||||||
| FLU | 4 (8.9) | 16 (35.6) | 25 (55.5) | x | x | |||||||
| TER | x | x | x | 4 (8.9) | 21 (46.7) | 20 (44.4) | ||||||
| ITR | x | x | x | |||||||||
| Tr (7) | GRI | x | x | 2 (28.6) | 4 (57.1) | 1 (14.3) | 0 | |||||
| FLU | 2 (28.6) | 5 (71.4) | x | x | ||||||||
| TER | x | x | x | 2 (28.6) | 2 (28.6) | 2 (28.6) | ||||||
| ITR | x | x | x | x | ||||||||
| Ng (6) | GRI | x | x | |||||||||
| FLU | 1 (16.7) | 1 (16.7) | 4 (66.6) | x | x | |||||||
| TER | x | x | x | |||||||||
| ITR | x | x | x | x | ||||||||
| Mc (2) | GRI | x | x | |||||||||
| FLU | 1 (50) | 1 (50) | x | x | ||||||||
| TER | x | x | x | |||||||||
| ITR | x | x | x | x | ||||||||
Trichophyton mentagrophytes species complex -Tm, Trichophyton rubrum-Tr, Nannizzia gypsum-Ng, Microsporum canis-Mc, MIC-minimum inhibitory concentration, CLSI-Clinical and Laboratory Standards Institute, GRI-Griseofulvin, FLU-Fluconazole, TER-Terbinafine, ITR-Itraconazole, X- indicates the antifungal concentration not tested. One isolate of T. rubrum was not assessed for terbinafine due to technical error
Table 2.
In vitro activities of antifungal agents against dermatophyte isolates, isolated from chronic and recurrent dermatophytosis patients
| Dermatophyte | Antifungal agent | MIC range (µg/mL) | MIC 50 (µg/mL) | MIC 90 (µg/mL) |
|---|---|---|---|---|
| T. mentagrophytes species complex | Griseofulvin | 0.125-0.5 | 0.25 | 0.5 |
| Fluconazole | 8-64 | 16 | 64 | |
| Terbinafine | 0.06-0.25 | 0.125 | 0.125 | |
| T. rubrum | Griseofulvin | 0.125-0.5 | 0.25 | - |
| Fluconazole | 8-64 | 8 | - | |
| Terbinafine | 0.06-0.25 | 0.125 | 0.25 | |
| N. gypsea | Griseofulvin | - | - | - |
| Fluconazole | 2-64 | - | - | |
| Terbinafine | - | - | - | |
| M. canis | Griseofulvin | - | - | - |
| Fluconazole | 4-64 | - | - | |
| Terbinafine | - | - | - |
MIC=Minimum inhibitory concentration. MIC50=MIC value at which growth was inhibited in 50% of isolates. MIC90=MIC value at which growth was inhibited in 90% of isolates
Discussion
We isolated T. mentagrophytes species complex, T. rubrum, N. gypsea, and M. canis from patients with chronic and recurrent dermatophytosis across rural and urban areas of Karnataka and Northern Kerala. The reasons for their chronic and recurrent nature may be multifactorial. Drug-related factors, including the susceptibility of the agent to the antifungal drug, the patient’s compliance with treatment, and the quality of the medication, seem to play key roles in the successful treatment outcome.[11] T. mentagrophytes was the most frequently isolated dermatophyte as seen in other studies. Studies have inferred that factors such as fomite transmission, untreated family members, poor hygiene, and other social factors may contribute to the occurrence of chronic dermatophytosis.[11,12] There has been an epidemiological shift from T. rubrum to T. mentagrophytes species complex as the primary causative agent in India.[11,13-15]
AFST for dermatophytes lacks standardization compared to other fungal agents or bacteria. While CLSI guidelines are commonly followed, specific breakpoints for dermatophytes are not widely established. In this study, all isolates depicted MIC values of ≤0.125 μg/mL for itraconazole, indicating favorable sensitivity to the lowest tested MIC value, and was consistent with other studies.[16,17,18] Itraconazole, a triazole antifungal, is effective against dermatophytes and is considered the first-line for recalcitrant dermatophytosis in India.[3] Failure of itraconazole therapy is rare, typically attributed to factors like severe disease or treatment compliance rather than antifungal resistance.[16]
Dermatophytes typically show low MIC values for azoles, except for fluconazole, where some authors report higher values of ≥16 μg/mL.[19] In our analysis, elevated MIC values against fluconazole (≥16 μg/mL) were observed in 20 isolates (80%) of T. mentagrophytes species complex, two isolates (28.5%) of T. rubrum, and one each of N. gypsea and M. canis. Our findings align with prior studies, reporting high MIC ranges for T. mentagrophytes species complex and T. rubrum.[20,21]
The MIC range for terbinafine was 0.06–0.25 μg/mL, both for T. mentagrophytes species complex and T. rubrum. Although recent reports indicate high terbinafine MIC values account for resistance, lower resistance rates in South India (16%) compared to other regions (75% or more) support our findings.[22] Our patients were from South India, so we justify our findings.
Most dermatophytes had MIC values of ≤1 μg/mL against griseofulvin. Our study observed MIC values of 0.25 μg/mL for approximately half of T. mentagrophytes species complex (53.3%) and T. rubrum (57.1%) isolates. In general, our isolates showed slightly lower MICs for griseofulvin compared to other studies.[19,21,23]
MIC 50 and MIC 90 values, representing the lowest concentrations of antifungal inhibiting 50% and 90% of isolates, respectively, were calculated only for T. mentagrophytes species complex and T. rubrum due to the small number of other isolates. MIC 50 and MIC 90 values were close to or lower than those reported in an Indian study.[24] However, terbinafine displayed MIC 50 and MIC 90 values of 0.125 μg/mL and 0.25 μg/mL for T. rubrum, in concordance with the aforementioned study, indicating susceptibility of dermatophytes across different regions may vary.
Limitations
A notable limitation is the absence of genotyping of isolate, necessitating cautious interpretation of the results. Nevertheless, itraconazole and terbinafine displayed favorable AFST profiles against all four isolates, suggesting their potential as first-line oral antifungals, as recommended in the Indian consensus guideline.[3] The sample size of isolates was small, except for T. mentagrophytes species complex, reflecting a significant epidemiological shift where T. mentagrophytes species complex has supplanted other agents as the predominant isolate.[25] We have not compared the results with studies where sequence-based identification to species level was done.[8,12]
Conclusion
T. mentagrophytes species complex is the most common cause of clinically diagnosed chronic and recurrent dermatophytosis lesions. It is heartening to notice that there is no in vitro resistance to itraconazole for any of the four isolated agents. Terbinafine resistance might not be consistent across India, with lower rates observed in Southern regions. However, there’s a possibility that it could eventually become a pan-Indian phenomenon. Periodic AFST and in vitro susceptibility testing across different regions of India are necessary to establish epidemiological cutoff values, aiding in the development of dermatophytosis management guidelines.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
We acknowledge the Indian Council of Medical Research (ICMR-AMR/Fellowship/9/2020 - ECD-II) for providing a Senior Research Fellowship to Mrs. Nikhitha Amin. We thank Dr. Anupma Jyoti Kindo, Professor and Head of Microbiology, Sri Ramachandra University, Chennai, for providing training to Mrs. Nikhitha Amin and Yenepoya Research Centre for allowing us to perform the experiments in the research laboratories.
Funding Statement
Nil.
References
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