A descriptive study of antifungal susceptibility of the dermatophytes isolates by microbroth dilution technique

INTRODUCTION

Dermatophytes are fungi that infect the superficial epidermis, damaging keratinized tissues of the skin, hair, and nails. These infections, known as ringworm or tinea, are classified by site: tinea pedis (feet), tinea capitis (scalp), tinea cruris (groin), tinea corporis (body), and tinea barbae (beard). In addition, a severe inflammatory form of tinea capitis is Favus, which occurs on the scalp. Non-dermatophyte fungi can cause similar skin infections, collectively termed dermatomycoses.^[1] Studies have revealed that such superficial infections caused by dermatophytes are responsible for approximately 90% of fungal skin infections worldwide. The dermatophytes can rapidly spread and grow on the superficial skin layers despite not being an integral part of the skin environment. This is because these fungi can thrive using keratin as a nutrient source.^[2] A typical “tinea” infection appears as a circular or annular lesion that is erythematous in nature, with the border comprising scales that grow in a centrifugal pattern. Infections such as eczema, psoriasis, atopic dermatitis, contact dermatitis, seborrheic dermatitis, and candida intertrigo can be mistaken for dermatophytosis.^[3] It is essential that the correct diagnosis of the type of dermatophyte infections, typically at skin and nail sites, be corroborated with clinical findings, microscopy, and fungal culture.

Dermatophytosis is frequently treated with a few antifungal medications; initially, griseofulvin was the only systemic antifungal medication that was authorized. Studies have revealed that new systemic and topical medications have been introduced into clinical practice to effectively treat dermatophytic conditions. These medications include terbinafine (allylamines), itraconazole and fluconazole (triazoles), naftifine (allylamines), and ciclopirox olamine (pyridine). In addition to the abundance of antimicrobial medicines, some researchers have noted that the mycosis-related dermatophyte resistance to the agent may be the reason for treatment failure.^[4]

This study aimed to identify the wide range of dermatophytes that can cause skin, hair, and nail infections in the patient population in a tertiary care hospital in Maharashtra. This was accompanied by antifungal susceptibility testing, which would help clinicians identify antifungal agents selectively for the prompt and corrective treatment of a particular type of fungal infection.

We used the standard microbroth dilution technique for testing antifungal susceptibility against the selected drugs, as this method is standardized and widely accepted. For our study, we aimed to determine the susceptibility of dermatophytes against clotrimazole, fluconazole, and terbinafine, which are the most commonly used antifungal drugs.^[1,5]

Since there is a notable rise in the prevalence of dermatophytosis, which is chronic, drug-resistant, and of repetitive type, particularly in a tropical country like India, it is imperative to understand and implement the minimum inhibitory concentration (MIC)-based microbroth dilution technique for antifungal susceptibility testing for dermatophytes in hospitals and clinical laboratories. Often, cases with increased severity of infections and a poorly managed treatment protocol are encountered. The microbroth dilution, as per Clinical and Laboratory Standards Institute (CLSI) M38-A2 or EUCAST protocols, is the gold standard for determining the MIC of antifungal drugs against dermatophytes. The results thus obtained are accurate and clinically acceptable. This is extremely essential as the dermatophyte species are continuously evolving and showing a varied range of susceptibility. Correct and precise MIC data not only helps clinicians prescribe the effective antifungal agent and dosage, but it also improves patient recovery and reduces any delay in correct drug advice. This technique is also useful for detecting any region-specific resistance patterns and for antifungal stewardship. Hence, with the help of our study, we recommend that in hospitals and laboratories with high patient load, the MIC-based microbroth dilution technique be adopted for accurate testing methodology and managing resistance.

MATERIALS AND METHODS

This was a descriptive study conducted over the duration of 24 months from June 2022 to May 2024 in the Department of Microbiology at a tertiary care hospital in Maharashtra. A total of 178 patients who were clinically suspected of dermatophytosis were included in the study. Samples (nail clippings, skin scrapings, and hair samples) received in the microbiology laboratory during the study period were subjected to culture and antifungal susceptibility.

Approval from the Institutional Ethics Committee was obtained.

RESULTS

A total of 178 clinically diagnosed cases of dermatophytosis were subjected to this study. Out of 178 samples, there were 165 skin scrapings, ten nail clippings, and three hair samples. The ratio of male: female occurrence of clinically diagnosed dermatophytosis was 1.2:1. Maximum frequency of dermatophyte isolates was observed in the age group of 18-27 years (23%). Out of the 165 skin scraping samples received, 67 patients were clinically diagnosed with tinea corporis, making it the most frequent dermatophyte infection, accounting for 40.6% of cases. There were 66 (37.1%) culture-positive samples, of which growth occurred in 63 skin scraping samples and three nail clipping samples. The hair samples, however, did not show any growth. Genus Trichophyton was found to be predominant (92.5%), followed by Microsporum (4.5%) and Epidermophyton (3%). Trichophyton mentagrophytes was observed to be the most predominant isolate from skin (57.1%) and nail (66.7%). Trichophyton rubrum was the second most common dermatophyte isolated from skin (35%) and nail (33.3%), respectively [Table 1]. In tinea corporis, T. mentagrophytes was the most common species isolated (58.6%). A similar finding was noted in tinea cruris, where isolates of T. mentagrophytes were in maximum numbers (56.5%), followed by T. rubrum (34.7%). Microsporum gypseum was isolated from samples of tinea corporis and tinea cruris by 6.4% and 4.3% respectively. Epidermophyton floccosum was isolated from tinea corporis (50%) and tinea cruris (50%). In tinea unguium, T. mentagrophytes was the maximum isolated species (66.7%), followed by T. rubrum (33.3%). In tinea faciei, 50% of isolates were T. mentagrophytes and 50% were T. rubrum. The Lactophenol cotton blue (LPCB) mounts of the isolates in high power (40x) have been shown in Figures 1a-d.

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Figure 1:

(a) Lactophenol cotton blue (LPCB) of Trichophyton mentagrophytes showing clusters of microconidia with cigar-shaped macroconidia (red arrows) under 40× (up to down). (b) LPCB of T. rubrum showing tear-drop microconidia with “bird on fence” appearance and pencil-shaped macroconidia (red arrows) under 40× (up to down). (c) LPCB of Microsporum gypseum showing thin-walled macroconidia with 4-6 (red arrows) septae under 40×. (d) LPCB of Epidermophyton floccosum showing club-shaped macroconidia (red arrows) under 40×. (e) Microtiter plate showing antifungal susceptibility testing (AFST) by microbroth dilution technique performed for fluconazole. (a-d) Scale bar=40 μm, 40X magnification.

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DISCUSSION

Over the past 20 years, there has been a notable rise in infections caused by dermatophytes and other fungi. Prolonged use of antifungal drugs can lead to acquired resistance in previously susceptible fungal strains.

This underscores the need for a standardized method to assess the susceptibility of the dermatophytes to antifungal drugs. The growing variety of available antifungal medications further highlights this necessity. Such a reference method would enable clinicians to make informed decisions about the most effective treatment for dermatophyte infections. This study focused primarily on evaluating the in vitro susceptibility of clinical isolates of dermatophytes to various antifungal agents.^[6,8,10]

The total sample size was 178, of these, 98 (55.05%) were male and 80 (44.95%) were female. Kanwar et al.,^[11] in 2001, showed that males were found to be affected more than females at 53% and 47%, respectively.^[11] Dermatophytosis was most common in the age group of 18-27 years (23%). Dermatophytosis is found to be common in the age group of 21-30 years in the studies done by Kaur et al.^[12] (23.3%) and Hazarika et al. (32%).^[13]

In this study, the most frequently diagnosed dermatophytosis is found to be tinea corporis with 67 cases (40.6%) followed by tinea cruris, 42 cases (25.4%). Bhatia and Sharma,^[7] Kumar et al., and Ghosh et al.^[14,15] stated similar findings of higher tinea corporis cases with 39.1%, 70%, and 58%, respectively, which is comparable to the study.^[7,14,15] There were 66 (37.1%) dermatophytes isolated from a total of 178 samples; nondermatophyte fungi and other contaminants grew in 59 (33.2%) of the specimens.

Potassium hydroxide (KOH) preparation is a widely used, simple, and effective diagnostic method for detecting fungal elements in clinical samples.^[14,16] In KOH examination of the samples, 49 (74.2%) were found to be KOH positive and culture positive. In the study done by Jain et al.,^[17] 72.5% of KOH and culture positivity was observed in their study.^[17] Out of 66 isolates of dermatophytes, 61 (92.5%) belonged to the Trichophyton spp., of which 38 (57.5%) were predominantly T. mentagrophytes, followed by 23 (35%) isolates of T. rubrum [Table 1]. In the study done by Bhatia and Sharma, T. mentagrophytes was the main isolate (98.6%).^[7] A similar result is seen in the study of Nenoff et al., with maximum occurrence of T. mentagrophytes by 93.21%.^[18] These studies are comparable to our current study. There appears to have been a shift in the epidemiology of dermatophytes in India in recent times. Even though T. rubrum has been identified as the most prevalent organism in India by numerous studies, the prevalence is far lower than it was in the past. T. mentagrophytes has surfaced as the co-dominant pathogen in each of our investigations, exhibiting a higher incidence than previously observed.^[19] Verma and Madhu state that there has been a significant shift in the prevalence and clinical manifestations of superficial dermatophytosis in India from the T. mentagrophytes to T. rubrum as the dominating species in just 10 years.^[20] Lakshmanan et al.^[21] said that geographical location, community trends, and socioeconomic circumstances all influence the distribution of dermatophytosis and its etiological agents.^[21] Kumar et al. also observed a decrease in the incidence of T. rubrum’s downy form.^[14] Three isolates (4.5%) of M. gypseum and two isolates (3%) of E. floccosum were found in this study. M. gypseum was at 1.8% in a study done by Kaur et al.^[22] The study done by Lakshmanan et al., M. gyseum was found to be 3.2% which is in concordance with this study.^[21] Ramaraj et al.^[23] showed a lesser occurrence of E. floccosum than the present study, that is, 0.6%.^[23] However, Rathod et al.,^[24] in their study, found the occurrence of E. floccosum at 2.38%.^[24]

T. mentagrophytes (36.3%) was predominant, which is consistent with the observations of Jamuna et al.,^[25] with T. mentagrophytes as the most isolated species (50.8%).^[25] This could be because the majority of people in this age range engage in outdoor activities, which puts them at risk of contracting infections from the environment.^[26,27] In our study, the P > 0.05 between the sexes indicated that both sexes are equally infected. Each of the four species of dermatophytes that were isolated for this investigation had the ability to impact males and females independently.^[19,21]

In our study for T. mentagrophytes, the MIC₅₀ and MIC₉₀ values for fluconazole were 2 mg/mL and 8 mg/mL [Table 2], respectively, closely aligning with Dhayagude findings of 2 mg/mL and 4 mg/mL.^[6] While our MIC₉₀ for fluconazole was slightly higher, the clotrimazole MIC₅₀ and MIC₉₀ of 0.25 mg/mL and 1 mg/mL, respectively [Table 3], were consistent with those reported by Fernandes et al., i.e., 0.25 mg/mL and 1 mg/mL, respectively.^[10] For terbinafine, our MIC₉₀ agreed with Fernandes et al.’s^[10] results, that is, 0.06 mg/mL, and our MIC₅₀ of 0.015 mg/mL [Table 4] was slightly lower than Indira’s value of 0.06 mg/mL.^[10,28] For T. rubrum, the MIC₅₀ of fluconazole was determined to be 2 mg/mL [Table 2], which is notably lower than the MIC50 of 8 mg/mL reported by Dhayagude^[6] The MIC₉₀ of 8 mg/mL in our study [Table 2] aligns with the finding of Dhayagure^[6] Furthermore, our results for clotrimazole’s MIC₅₀ and MIC₉₀ of 0.125 mg/mL and 0.25 mg/mL, respectively [Table 3], were consistent with those observed by Fernandes et al.^[10] Regarding terbinafine, the MIC50 of 0.03 mg/mL in our study [Table 4] is comparable to that of Fernandes et al.^[10] However, we observed a slightly higher MIC₉₀ for terbinafine at 0.06 mg/mL [Table 4], compared to 0.04 mg/mL reported by Indira.^[28] The present study’s findings on antifungal susceptibility are largely consistent with previous research. For M. gypseum, MIC₅₀ and MIC₉₀ values of 4 mg/mL and 32 mg/mL for fluconazole [Table 3], mirroring those reported by Suganthi et al.^[29] Similarly, our results for clotrimazole of MIC₅₀ at 0.5 mg/mL and MIC₉₀ at 1 mg/mL [Table 3] align with those of Fernandes et al.^[10] For terbinafine, MIC₉₀ of 0.06 mg/mL [Table 4] matches those of Fernandes et al.^[10] and Suganthi et al.,^[29] and our MIC₅₀ of 0.015 mg/mL [Table 4] is concordant with Suganthi et al. and Kumar et al.^[10,29,30] For E. flocossum, MIC₅₀ for fluconazole was 16 mg/mL [Table 2], which aligns with the findings of Afshari et al.^[31] However, our MIC₉₀ of 64 mg/mL [Table 2] is slightly elevated compared to their MIC₉₀ of 32 mg/mL. For clotrimazole, our MIC₅₀ of 0.25 mg/mL [Table 3] is consistent with the studies by Fernandes et al.^[10] Yet, our MIC₉₀ of 0.5 mg/mL [Table 3] is slightly higher than theirs. Finally, our MIC₅₀ and MIC₉₀ values for terbinafine with 0.03 mg/mL and 0.06 mg/mL, respectively, are in agreement with the results reported by Fernandes et al.^[10]

In the overall antifungal susceptibility tests conducted on 66 dermatophyte isolates, terbinafine demonstrated the lowest MIC range, spanning from 0.015 to 0.06 mg/mL. Clotrimazole followed with a MIC range of 0.125-1 mg/mL. The highest MIC range was observed with fluconazole, at 2-64 mg/mL. The MIC₅₀ values mirrored this trend, with terbinafine exhibiting the lowest at 0.015 mg/mL, followed by clotrimazole at 0.125 mg/mL.

Fluconazole recorded the highest MIC₅₀ at 2 mg/mL. Similarly, the MIC₉₀ for terbinafine was the lowest at 0.06 mg/mL followed by clotrimazole at 1 mg/mL. Fluconazole again displayed the highest MIC₉₀, reaching 64 mg/mL [Table 5].

The most sensitive antifungal agent found in this study is terbinafine, followed by clotrimazole. The highest MIC was found in the case of fluconazole. It is similar to the study done by Indira and Ganesan et al.,^[32] where terbinafine was found to be the most sensitive drug against dermatophytosis.^[28,32] Recent years have seen several investigations into the in vitro susceptibility of dermatophytes, with notably variable results. It is likely that significant methodological variations between the laboratories are the cause of this diversity. Antifungal susceptibility testing for dermatophytes remains technically challenging due to the inherent morphological characteristics of these fungi. Some species, particularly those adapted to human hosts (anthropophilic), exhibit poor sporulation, a challenge further exacerbated by repeated subculturing. While various methods for testing dermatophytes’ susceptibility to antifungals have been assessed, results have shown considerable inconsistency. Discrepancies in the MIC of antifungal agents can be attributed to various factors, including technical challenges, particularly concerning parameters such as inoculum size, incubation duration, media interference, insolubility, temperature, and endpoint criteria, all of which can significantly influence MIC determinations.^[10,11] The microdilution assay for antifungal susceptibility testing provides a practical and reproducible method. Our research on the dermatophytes’ susceptibility to antifungals is expected to be useful for managing patients who are clinically resistant to therapy, as well as for examining the dermatophyte species’ in vitro resistance. Emerging dermatophyte species like Trichophyton indotineae are morphologically very similar to T. mentagrophytes (complex) and require genomic sequencing for identification.^[33] This was considered a limitation. We plan to perform genomic sequencing of such species in the next phase of the study.

CONCLUSIONS

Analysis of antifungal susceptibility tests conducted using the microbroth dilution technique by the CLSI M38-A2 has shown significant findings establishing the efficacies of various antifungal agents against the tested fungal strains. Among the antifungal agents tested, terbinafine emerged as the most sensitive antifungal agent, exhibiting the lowest MIC.

Clotrimazole exhibited the next lowest MIC, emerging as a viable option; its sensitivity is slightly less pronounced compared to terbinafine, as is evident from our study results as an antifungal agent.

Fluconazole displayed the highest MIC, indicating reduced sensitivity, signifying reduced susceptibility of the fungal strains to fluconazole. The higher MIC value indicated that fluconazole required a higher concentration to achieve the same level of fungal growth inhibition of the chosen fungal strains.

Incorporating AFST into routine laboratory protocols is essential to detect local resistance patterns and prevent the indiscriminate use of antifungal agents. This practice enables clinicians to initiate appropriate antifungal therapy, contributing to improved patient outcomes and supporting public health efforts to reduce the burden of dermatophytosis.