In vitro MIC comparison of a test itraconazole brand versus 4 brands of itraconazole against clinical isolates of Trichophyton mentagrophytes and Trichophyton rubrum using broth dilution method and disc diffusion method

Dermatophytosis is one of the most common cutaneous fungal infections in India. Despite, quite a few numbers of antifungal agents available for treatment, the resistance to antifungals by dermatophytes is increasing day by day as seen by doctors in clinical practice. Itraconazole has shown good results in the treatment of dermatophytosis at doses of 100 mg once a day for 2 weeks and with 200 mg once a day for 7 days. However, there are multiple itraconazole brands available in India. In this study, five different itraconazole brands were evaluated in vitro for MIC values against clinical isolates of Trichophyton mentagrophytes and Trichophyton rubrum by two different methods i.e. broth dilution and disc diffusion tests. It was found that the Reference brand of itraconazole and the test brand of Itraconazole showed least MIC values as compared to other available brands. © 2020 Published by Innovative Publication. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by/4.0/)


Introduction
Infection of the hair, skin, or nails caused by a fungus dermatophyte is called as dermatophytosis. Amongst dermatophytes, the most common genus is Trichophyton followed by Microsporum or Epidermophyton genera. Tinea capitis, tinea pedis, and onychomycosis are common dermatologic diseases caused by dermatophytes. 1 Trichophyton rubrum and Trichophyton mentagrophytes, which cause infections of skin and nails, are two of the most commonly isolated dermatophytes. 2 A large number of safe and effective antifungal agents are available for the treatment of dermatophytosis. 3 Antifungal agents such as triazoles (itraconazole, fluconazole), imidazole (ketoconazole), allylamine (terbinafine) and griseofulvin have been reported to have substantial activity in dermatophytosis. 4 It has been observed recently that there has been widespread resistance to various antifungal agents used

Specimen
The strains of T. mentagrophytes and T. rubrum (50 of each) used in this study were obtained from skin and nails of patients with dermatophytosis and onychomycosis. The isolates were identified by routine mycological procedures and were maintained in sterile distilled water at 4 0 C until tests were performed.

Itraconazole dilutions
The Itraconazole was obtained from all 5 manufacturers including innovator. Drugs were dissolved in 100% dimethyl sulfoxide (Sigma-Aldrich). They were subsequently prepared as stock solution and serial twofold dilutions were performed.

Broth dilution method
The Dermatophytes were sub-cultured on Sabouraud Dextrose Agar & incubated at 28 • C for 9-10 days to enhance sporulation. The growth was harvested in sterile saline & the conidial and hyphal suspension was adjusted to 1x10 6 /ml using a haemocytometer. This suspension is used as an inoculum for the broth dilution method. For the broth the dilution sterile Sabouraud Dextrose broth supplemented with 2% Glutamine was used. The stock solution with 20 µg/ml (w/v) of each formulation was prepared. Stock solution was then used to perform 2fold serial dilution to achieve the concentration range of 10 µg/ml to 0.005 µg/ml (v/v). Tubes were then inoculated with 0.1 ml of inoculum and incubated at 28 • C for 9-10 days. The medium control and Positive Growth control of inoculum was also maintained along with the test sets. After the completion of incubation period the tubes were observed for turbidity.

Agar Disc Diffusion Method
The Dermatophytes were sub-cultured on Sabouraud Dextrose Agar & incubated at 28 • C for 9-10 days to enhance sporulation. The growth was harvested in sterile saline & the conidial and hyphal suspension was adjusted to 1x10 6 /ml using a haemocytometer. This suspension is used as an inoculum for the Agar Disk Diffusion method.
The Sabouraud Dextrose Agar supplemented with the 2% Glutamine were used to perform the test. The 10 µg/µl, 2.5 µg/µl, 0.18 µg/µl, 0.04 µg/µl and 0.01 µg/µl concentrations of formulations were made in filter sterile DMSO. 5 mm filter paper disks were made, autoclaved and dried. Sabouraud agar plates with 2% Glutamine were inoculated with the dermatophytes using sterile swab and formulation disc were placed on the agar plates. The plates were sealed with parafilm and kept for incubation at 28 • C for 9-10 days. After the completion of incubation plates were observed for zone of inhibition. The outer diameter of each such zone measured and results were interpreted accordingly.

Endpoint determination
For broth dilution method, the tube with least concentration of formulation showing no turbidity was considered as MIC. The medium control tube did not show any turbidity while Positive Growth Control tubes exhibited turbidity indicating growth of the inoculum.

Results
MICs of antifungal agents for dermatophytes isolates could be determined after four days for T. mentagrophytes and five days for T. rubrum when incubated at 28 0 C. ummarizes the MIC ranges for all brands against the isolates ofT. mentagrophytes and ummarises MIC ranges againstT. rubrum.  For agar disc diffusion method, diameter of zone of inhibition are illustrated in Tables 3 and 4 for T. mentagrophytesandT. rubrum respectively.

Discussion
Dermatophyte infections are widespread and cause significant distress to the patients socially, emotionally and financially. Resistant dermatophytosis is fast emerging as a challenge for dermatologists in India. T. mentagrophytes is one of the most commonly isolated organisms. This has been the most frequently isolated organism in some reports from India, although in some studies of tinea corporis and T cruris, T. rubrum has been more frequently isolated. 6  Widespread resistance to conventional doses of antifungals with increasing clinical failure rates warrants the search for an effective first-line antifungal drug that brings about rapid clinical and mycological cure in tinea corporis and tinea cruris. Itraconazole is a triazole antifungal drug which is also increasingly being used as a first-line drug for tinea corporis and tinea cruris. In a study, Itraconazole was the most effective antifungal agent against T. mentagrophytes, T. rubrum. 7 The taxonomy of dermatophytes is evolving with increased use of molecular techniques and T. mentagrophytes and T. rubrum are now considered as a species complex with many species defined within each species. 8 In this study, a CLSI protocol (standard M38-A) adapted was followed to determine the MIC values of five different brands of itraconazole currently employed for management of dermatophytosis in India. The determination of the in vitro susceptibility may prove helpful to predict the ability of a given antifungal agent to eradicate dermatophytes. Although a reference method for dermatophytes, is not available, a good correlation between the in vitro data, using broth microdilution method or agar disc diffusion method, and clinical outcome has been demonstrated. 9 With broth dilution method, Reference brand of itraconazole showed least MIC against T. mentagrophytes (0.18 µg/ml) and T. rubrum (0.35 µg/ml). The test Itraconazole Formulation was found to be the closest to Reference itraconazole brand with respect to MIC concentration among other four Formulations. The MIC of test Itraconazole brand for T. mentagrophytes and T. rubrum were 0.35 µg/ml and 1.25 µg/ml respectively. In case of agar disc diffusion method, among 5 tested formulations the zone of inhibition pattern of Test Itraconazole brand and Reference itraconazole brand found to be similar against T. rubrum and T. mentagrophytes.

Conclusion
This is the first of its kind study where different antifungal preparations of same molecule i.e. itraconazole were evaluated using two different in vitro methods. It can be concluded that MIC values for Test Itraconazole brand and reference itraconazole brands were least as compared to other generic brands when assessed using in vitro susceptibility testing including broth solution and agar disc diffusion methods against the clinical isolated of T. rubrum and T. mentagrophytes

Acknowledgements
We acknowledge our sincere gratitude to Glenmark Pharmaceuticals Limited for help in data analysis and preparation of the manuscript.

Source of funding
This study was carried out with the grant from Glenmark Pharmaceuticals Limited, as an unrestricted educational grant.