The in vitro Activity of Echinocandins Against Clinical Trichophyton rubrum Isolates and Review of the Susceptibility of T. rubrum to Echinocandins Worldwide

Introduction

Dermatomycosis is a prevalent superficial fungal infection primarily affecting keratinized tissues, including the skin, hair, and nails, with a remarkably high incidence rate of 20–25%.¹ Dermatophyte species are the most common causative agents of dermatomycosis. Of the more than 40 dermatophyte species infecting humans, Trichophyton rubrum is the most common species,² although there has been a rise in infections caused by the Trichophyton indotineae in Trichophyton mentagrophytes/Trichophyton interdigitale complex in India.^3,4 Generally, dermatophytosis is easily cured with a wide range of topical and oral antifungal drugs. Azoles and allylamines are the primary recommended topical and systemic agents for treating T. rubrum infections. Terbinafine, renowned for its clinical efficacy, is the most commonly prescribed drug against Trichophyton species due to its fungicidal properties.⁵ However, since the first reported case of T. rubrum resistance to terbinafine in 2003,⁶ reports of T. rubrum resistance to antifungal agents have been increasing in the Americas, Europe, and Asia.^7–9 Several studies have demonstrated that T. rubrum can develop resistance to terbinafine, azoles, and amorolfine after prolonged exposure to sub-inhibitory concentrations of these drugs.^6–12 An epidemiological study conducted in India reported a remarkably high frequency of terbinafine-resistant T. rubrum strains, reaching 44% (8 out of 18 isolates).¹³ A review encompassing 26 global studies on T. rubrum susceptibility revealed that 15 studies (57.69%) exhibited a terbinafine geometric mean above 0.03 mg/L (the epidemiological cut-off value), indicating the presence of non-wildtype T. rubrum strains with potential pandemic characteristics.¹⁴ In India, T. indotineae in T. mentagrophytes/T. interdigitale complex replaces T. rubrum as the most common skin infection in the last decade.¹⁵ Notably, the highest resistance rates of the T. indotineae have been observed in northern India, with a terbinafine resistance rate of 76% (MIC ≥0.5 mg/L).¹³ T. indotineae appears to be spreading towards Europe and other Asian countries.¹⁵ Therefore, the development of new antifungal drug classes is crucial in overcoming the existing and future challenges in managing dermatophyte infections.

Echinocandins are lipopeptides derived from the fermentation broths of various fungi, which have been synthetically modified. These compounds act by inhibiting the synthesis of cell wall β-1,3-glucan.¹⁶ Echinocandins have been approved for invasive aspergillosis and candidiasis. Echinocandins generally exhibit fungistatic and fungicidal activities against Aspergillus and Candida species, respectively.^17,18 Echinocandins have also shown in vitro activity against dermatophytes but reports on clinical use are lacking;¹⁹ however, the data on the antifungal efficacy of echinocandins to dermatophytes in vitro are limited.^19–23 In this study, we aimed to evaluate the potency of echinocandins (anidulafungin, caspofungin and micafungin) against a large set of clinical T. rubrum isolates collected from China and review the existing literature on in vitro susceptibility of echinocandins against clinical isolates of T. rubrum worldwide.

Materials and Methods

T. rubrum Isolates and Identification

A total of 73 clinical isolates of Trichophyton rubrum were collected from patients diagnosed with dermatophytosis between 2014 and 2018 at Huashan Hospital, Fudan University in Shanghai, China. The diagnosis of dermatophytosis was established through a combination of clinical presentation and mycological examination:²⁴ (a) the presence of skin lesions indicative of dermatophytosis; (b) positive direct microscopy demonstrating the presence of hyphae in the specimen; and (c) positive culture of dermatophytes.

The 73 clinical T. rubrum isolates were identified using morphological features and subsequently confirmed by sequencing the ITS gene as described previously.²⁵ Isolates information and GenBank accession numbers for the generated ITS sequences of the 73 T. rubrum isolates are listed in Table S1.

Antifungal Susceptibility Testing

In vitro antifungal susceptibility testing of the 73 isolates of T. rubrum was performed according to the EUCAST E.DEF 9.3.1 (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_9_3_1_Mould_testing__definitive.pdf) and adjusted part of the parameters for dermatophytes. The isolates were subcultured on potato dextrose agar and incubated at 28°C for 7 days or longer to sporulate sufficiently. Cover colonies with sterile water supplemented with 0.1% Tween-20. Then, the culture was gently swabbed with a cotton tip and the conidia are transferred with a pipette to a sterile tube. After vortexed, the suspension was counted with a haemocytometer chamber and checked the no presence of hyphae. If a significant number of hyphae is detected, transfer the suspension to the filter to remove hyphae and yield a suspension composed of conidia. The suspension is then diluted with sterile distilled water to obtain a final working inoculum of 2–5 × 10⁵ CFU/mL. Anidulafungin and caspofungin were purchased from Sigma-Aldrich (MO, USA). Micafungin was bought from Toronto Research Chemicals (Toronto, Canada). The testing range for micafungin was 0.002 to 2 mg/L; for anidulafungin and caspofungin were both 0.016 to 16 mg/L. Vortex the inoculum suspension and inoculate each well of a microdilution plate with 100μL of the conidial suspension. Panels were incubated at 28°C for 4–5 days. MECs were first read at 4 days incubation. The final results were determined when the control well got sufficient growth. The MEC denotes the lowest echinocandin concentration at which the fungus displayed growth retardation and/or microscopic morphological changes in contrast to the control well. There are no clinical breakpoints available for all these drugs yet. Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as the quality control strains. The original antifungal susceptibility testing data are listed in Table S1.

Literature Review

Literature searching was carried out in databases including PubMed, Scopus, and Web of Science. We retrieved the papers using the terms “(Trichophyton rubrum) AND (echinocandins OR anidulafungin OR caspofungin OR micafungin)” in the title and abstract.

Results

Table 1 lists the MEC ranges, geometric mean (GM), modal MEC, and MEC distribution of three echinocandins against 73 clinical T. rubrum isolates tested in this study. Figure 1 shows the modal MEC and MEC distribution of three echinocandins against 73 clinical T. rubrum isolates tested in this study. The MEC ranges of micafungin, anidulafungin and caspofungin were 0.002–0.063 mg/L, 0.016–0.063 mg/L and 0.031–1 mg/L, respectively. Micafungin had the lowest modal MEC (0.008mg/L, n = 28) and MECs geometric mean (GM) (0.014 mg/L), followed by anidulafungin (modal MEC 0.016mg/L, n = 67, GM 0.018 mg/L) and caspofungin (modal MEC 0.5mg/L, n = 35, GM 0.308 mg/L) against all T. rubrum isolates tested.

Table 1 MEC Ranges, GM, Modal MEC, and MEC Distribution of 3 Echinocandins Against 73 Clinical T. rubrum Isolates from China

Figure 1 MEC distribution of 3 echinocandins against 73 clinical T. rubrum isolates from China.

The literature review resulted in 5 publications reporting the susceptibility of T. rubrum to echinocandins worldwide from 2013 to 2022^19–23 (Table 2, Figure 2). Table 2 summarizes the susceptibility data of T. rubrum isolates to echinocandins based on the 5 publications and the results of our study. Of all 6 studies, our study included the largest set of T. rubrum isolates (73 isolates) and 3 echinocandins (micafungin, anidulafungin, caspofungin), the rest of the studies tested sample sets varying from 7 to 43 isolates of T. rubrum, and 4 literatures involved only two echinocandins. The methods used in all 6 studies included Clinical and Laboratory Standards Institute (CLSI) M38-A2, European Committee on Antimicrobial Susceptibility Testing (EUCAST), modified EUCAST, and E-Test. The culture mediums for T. rubrum were mainly potato dextrose agar (PDA) and oatmeal agar (OA). Only the modified method in Rissleger’s study used Sabouraud −2% glucose agar (SAB) to get fragmented mycelium for inoculum preparation. The incubation temperature was 28°C in Aktas’s study using the E-test method.²⁰ Similarly, in our study, we utilized the modified EUCAST method and maintained the incubation temperature at 28°C. Rissleger’s study adopted a slightly higher temperature of 30°C while using the modified EUCAST method.²² In the remaining studies, the incubation temperature was set at 35°C. The incubation period varied from 2 to 9 days.

Table 2 Summarized Data of the 6 Studies on Susceptibility of T. rubrum to Echinocandins

Figure 2 Summarized data of 6 studies on antifungal susceptibility of T. rubrum to echinocandins.

Caspofungin is the most common echinocandin tested for the activity against T. rubrum in all 6 studies. MEC ranges and GM, MEC₉₀ values are variable in different studies, with the highest MEC range (1–8mg/L) in Badali et al’s study and the lowest MEC range (0.008–0.032mg/L) in Baghi et al’s study (Table 2, Figure 2).^21,23

Micafungin exhibited the lowest MEC value with MIC value relatively consistent (GM varying from 0.010 to 0.034 mg/L, MIC₉₀ from 0.016 to 0.06mg/L) among 4 studies in which Micafungin was included (Table 2, Figure 2).

Only 3 studies reported the activity of anidulafungin against T. rubrum. Generally, anidulafungin exhibited a similar MEC range (0.008–0.063mg/L) among 3 studies although small samples were tested in studies by Badali et al and Rissleger et al.^21,22 However, there are two diluted steps difference for MIC₉₀ value (0.016mg/L) of anidulafungin against T. rubrum in our results compared to those in the other two studies (MIC_90, 0.063mg/L) (Table 2, Figure 2).

Discussion

Echinocandins are a class of antifungal drugs with milder side effects compared to polyenes and azoles, since these agents only inhibit the fungal-specific metabolic pathway—β-(1,3)-glucan biosynthesis.²⁶ Caspofungin acetate was the first synthesized echinocandin and then approved for clinical use by the US Food and Drug Administration (FDA) in 2001, followed by micafungin in 2005 and anidulafungin in 2006. Because of the good drug safety, echinocandins were even approved for the treatment of children over 3 months of age and there is no need to adjust the dose in renal or hepatic insufficient patients.²⁷ The FDA has approved the indications of echinocandins in the empirical treatment of febrile neutropenia, Candida infections, invasive aspergillosis in refractory patients, or intolerant to other therapies.

However, the antifungal susceptibility profile of dermatophytes to echinocandins remains poorly examined. Among 5 literatures reviewed and our results (Table 2, Figure 2) in this study,^19–23 the activity of caspofungin against T. rubrum was tested in all 6 studies, whereas micafungin was tested in 4 studies, and anidulafungin only in 3 studies. Most studies are with a limited sample set of T. rubrum isolates (7–43 isolates) except our study with 73 isolates of T. rubrum.

Though there are no reference breakpoints of echinocandins to T. rubrum available at the moment, in our study, three echinocandins tested showed good activity against the 73 clinical T. rubrum isolates in this collection, while micafungin and anidulafungin showed a lowest modal MEC of 0.008 mg/L (n = 28) and 0.016 mg/L (n = 67), respectively, followed by caspofungin with modal MEC of 0.5 mg/L (n = 35) (Table 1, Figure 1). Micafungin and anidulafungin appeared more potent than caspofungin against T. rubrum, and similar results have been achieved in other studies.^19–23

Baghi et al²³ tested in vitro activity of caspofungin and anidulafungin against clinical dermatophyte isolates using CLSI M38A2, both echinocandins demonstrated potent activities against dermatophyte isolates, the MEC₅₀, MEC₉₀, and GM values of caspofungin and anidulafungin against 29 isolates of T. rubrum tested in the study by Baghi et al were similar to those reported in our study (Table 2, Figure 2). Badali et al²¹ presented in vitro susceptibility patterns of clinically important Trichophyton and Epidermophyton species against nine antifungal drugs including caspofungin and anidulafungin using CLSI M38A2, caspofungin exhibited higher MEC value (GM 1.67mg/L, MEC₉₀ 4mg/L) against Trichophyton and Epidermophyton species, the GM, MEC₉₀ of caspofungin against 13 T. rubrum isolates tested in the study were 1.53mg/L and 4 mg/L, respectively. Nevertheless, anidulafungin exhibited potent in vitro antifungal activity against all dermatophyte isolates (GM, 0.06mg/L) and excellent activity for T. rubrum as well (GM, 0.02mg/L). Bao et al¹⁹ tested the potency of micafungin and caspofungin against dermatophyte isolates from China using CLSI M38-A2. Both caspofungin and micafungin exhibit good in vitro antifungal activity in most dermatophytes compared with the traditional antifungal agents (terbinafine). The GM, MEC₉₀ values of micafungin and caspofungin to 30 isolates of T. rubrum tested are similar to those in our study. In addition, the E-test method was used to investigate the in vitro activity of caspofungin against 43 isolates of T. rubrum in Aktas’s study²⁰ and caspofungin exhibited as an active agent with a MEC range of 0.02–3mg/L for T. rubrum. Rissleger et al²² evaluated the modified EUCAST fragmented-mycelium inoculum method for in vitro susceptibility testing of dermatophytes and the activity of novel antifungal agents including 3 echinocandins. Consistency between the mycelial inoculum method and the CLSI broth microdilution method was high (93% to 100%). Echinocandins showed good activity against all tested dermatophytes and worked as the most effective agents against dermatophytes compared with other drugs like voriconazole, posaconazole, fluconazole, and amphotericin B. All the MEC values of caspofungin, micafungin and anidulafungin for T. rubrum isolates tested in the study are comparable to those in other studies (Table 2, Figure 2).

There are certain limitations in this study. As this work had been done before EUAST method for the susceptibility testing of dermatophytes was launched.²⁸ We followed the EUCAST E.DEF 9.3.1 edition with minor modification on incubation temperature and time for dermatophytes. Of note, the EUCAST method for dermatophytes²⁸ was mainly validated for terbinafine, voriconazole, itraconazole and amorolfine against T. rubrum and T. interdigitale, however, the susceptibility testing of dermatophytes to echinocandins is observed with MEC which is not included in the EUCAST method for dermatophytes yet.²⁸ The standard protocol on the susceptibility testing of dermatophytes to echinocandins is needed urgently.

Overall, micafungin and anidulafungin exhibited a low MEC range (both 0.008–0.063 mg/L) to T. rubrum which are similar among those studies,^19,21–23 relatively higher MEC values for caspofungin to T. rubrum isolates were observed in 5 studies despite different methods and culture mediums when compared with those for anidulafungin and micafungin (Table 2, Figure 2), which indicated that micafungin and anidulafungin have better activity than caspofungin against T. rubrum. Micafungin has stronger in vitro antifungal activity than caspofungin in our study and also other research¹⁹ (Table 2, Figure 2). Echinocandins generally exhibit fungistatic and fungicidal activities against Aspergillus and Candida species, respectively.²⁹ Comparing the in vitro potency of echinocandins to Aspergillus fumigatus, anidulafungin and micafungin were also the most active drugs against A. fumigatus in vitro with the lowest modal MICs/MECs (0.016mg/L, n = 61 and 0.031mg/L, n = 64, respectively), followed by caspofungin (0.25mg/L, n = 119) when compared with azoles and AmB in our previous study,³⁰ and similar results reported by our previous study.³¹

Conclusion

Our study provided evidence of the good potency of micafungin, anidulafungin and caspofungin against T. rubrum in vitro based on our results and review of the data published on the susceptibility of T. rubrum to echinocandins worldwide. These findings suggest that echinocandins could be considered as potential treatment options for recalcitrant dermatophytoses infected by resistant dermatophytes. However, further clinical investigations must be conducted for the in vivo efficacy of echinocandins for the treatment of dermatophytosis caused by T. rubrum.