COMPARISON OF THE ACTIVITIES OF FOUR ANTIFUNGAL AGENTS IN AN IN VITRO MODEL OF DERMATOPHYTE NAIL INFECTION

Background: Onychomycosis is a diffi cult condition to treat and cure rates are disappointing. Moreover fungicidal action of antifungal agents in NCCLS assays and their rapid accumulation in nails in vivo are not compatible with the duration of treatment. Aims: This study aimed to fi nd the effectiveness of 4 different antifungal agents in an in vitro model with some similarities to in vivo conditions. Materials and Methods: Strains of Trichophyton rubrum I-III, Trichophyton mentagrophytes (usual form), Trichophyton mentagrophytes 73, Epidermophyton Flucosom, Microsporum Canis, and Trichophyton Schoenleini which were isolated from the nails of patients, were hired. Inocula suspensions were prepared from 7 to 14 day-old cultures of dermatophytes. Antifungal agents including fl uconazole, ketoconazole, terbinafi ne, and griseofulvin were obtained as standard powders. For each antifungal agent, initial MIC was calculated by registering the optical density for 10 two-fold serially diluted forms which was incubated with diluted fungal suspensions with RPMI 1640. Human nail powder inoculated with different strains and incubated in RPMI 1640 and different concentrations of antifungal drugs for 4 weeks. Final MIC at different steps of 1, 2, 3 and 4 weeks were investigated. Results: The fi nal MIC that resulted from the incubation of dermatophytes with nail powder was much more than the initial which was concluded from conventional MIC assay. Terbinafi ne had the lowest rate of initial and fi nal MICs. Conclusion: The model described here may present more similar conditions to clinical fungal infections; therefore the results such as MIC may be more helpful for hiring the most effective antifungal agent.


Introduction
Onychomycosis (OM) is fungal infection of the toenails or the fi ngernails.It is not life-threatening, however it can cause pain, discomfort, and deformity and may have a signifi cant effect on the quality of life. 1 Half of all nail disorders are due to OM.Moreover, 30 percent of patients with a cutaneous fungal infection also have OM.Additionally, the incidence of OM has been increasing, on account of increasing in the incidence of diabetes mellitus, immunosuppression and increasing age. 2 Despite fungicidal effect of anti-fungal drugs (e.g.terbinafi ne) in National Committee for Clinical Laboratory Standards (NCCLS) assays and their rapid accumulation in nails in vivo, onychomycosis patients require prolonged treatment to be cured. 3Therefore, an in vitro model with some similarities with in vivo conditions might help us to assess this dilemma.In addition, since this infection is very common, especially in elderly patients, effective topical therapy would provide an outstanding treatment for such patients. 4om the Iran University of Medical Sciences, Tehran, 1 Gom Azad University, Gom, 2 Tehran University of Medical Sciences, Tehran, 3 Karaj Azad University, Karaj, Iran.Address correspondence to: Dr. Hossein Nowrozi, Department of Medical Mycology, Faculty of Health, Iran University of Medical Sciences, Hemmat Highway, Tehran, Iran.E-mail: nowrozi@usa.comcarefully with the tip of a Pasteur pipette.Heavy particles of the suspension were allowed to settle for 3 to 5 min.The fi nal inoculum size was adjusted with a spectrophotometer at a wavelength of 530 nm to a transmittance of 95%.These suspensions were diluted in RPMI 1640 test medium (1:50) to obtain a cell number ranging from 0.5 × 10 4 to 5 × 10 4 CFU/ml.Antifungal agents: They were obtained as standard powders.The powder of fl uconazole 99.7% (Pars Daru, Iran), ketoconazole 98.43% (Rooz Daru, Iran), terbinafi ne 99.6% (Behvarzan, Iran), and griseofulvin 99.3% (Daroopakhsh, Iran) were used in this study.
Stock solutions were prepared by dissolving and two-fold serially dilution of drugs in Methanol 0.1 percent, by using Broth microdilution method.10× dilutions were prepared for each drug Tested concentrations for fl uconazole ranged from 0.187-96.0μg/ml, and for ketoconazole, terbinafi ne and griseofulvin from 0.125-64.0μg/ml.Stock solutions were prepared by dissolving and two-fold serially dilution of drugs in Methanol 0.1 percent.

Determination of initial MIC:
For each fungal strain 13 tubes with the volume of 10 ml, were prepared.0.1 cc of each antifungal agent dilutions were added to tubes 1 to 10. Then 0.9 cc of diluted fungal suspensions with RPMI 1640 was added to each tube.The eleventh tube contained a 0.9 ml volume of inoculum suspension and a 0.1 ml volume of drugfree medium (To assess the inhibitory effect of medium on fungal growth).The twelfth tube (growth control) consisted of fungal suspensions with RPMI 1640.A sterility control (the thirteenth tube) was run in parallel by including a 1 ml volume of uninoculated, drug-free medium.
Tubes were incubated at 30°C for 48h.Growth control tubes were observed for the presence or absence of visible growth.When growth was visible, the growth in each tube was compared with that of the growth control tube.Optical density (OD) of each tube which was obtained from a spectrophotometer at a wavelength of 530 nm was used to fi nd the amount of reduction in turbidity as compared to that of the drug-free control tube.MIC at which 50% of the isolates are inhibited (MIC 50 ) was determined.
Preparation of nail powder: 5,10 Human nail Clippings from several healthy volunteers previously found free from fungi when examined in 10% KOH were used.The samples were cleaned with 70% ethanol, dried in a sterile Petri dish at 37°C, and ground with using a stainless steel peppermill.The powder was autoclaved at 121°C for 20 min.
Nail model culture: After fi nding the primary MIC for each microorganism, 6 tubes containing MIC tube, 2 tubes with higher dilution and 2 tubes with lower dilution, respectively plus another tube as the control tube were selected.Autoclaved nail powder was added, at approximately 10 mg per tube.0.9 cc of Inocula suspensions were added directly onto the nail powder.The tubes were left at 37°C for 10 days.After 10 days the mycelium were attached to nail particles which established the suffi ciency of the growth time.0.1 cc of antifungal agents were added to the culture according to the calculated MIC, and the cultures were returned to 37°C.Final MIC 4 weeks after incubation with nail powder was investigated.

Statistical analysis
Initial and fi nal MICs were assessed 2 times for each sample and presented as means.ANOVA besides Tukey test was hired to analyze MICs in each subgroup.Pearson correlation was used in order to fi nd any correlation between initial and fi nal MICs.Paired t-test was used for comparing between initial and fi nal MICs.

Results
Initial MICs [before culturing on nail powder] for each subgroup are presented in Table 1.
Grisofulvin and ketoconazole had the same rate of initial MICs, which were statistically different from fl uconazole with the highest rate of initial MIC and terbinafi ne with  2].
There was a direct correlation between initial and fi nal MICs which was statistically signifi cant (R = 0.95, P value = 0.0001).

Discussion
Griseofulvin and newer antifungal drugs such as ketoconazole, fl uconazole and terbinafi ne, are the major systemic drugs applied to treat onychomycosis. 11Griseofulvin was the fi rst systemic antifungal drug but nowadays it is not used in a widespread way. 12Ketoconazole was the fi rst orally active imidazole but it is known for its hepatotoxicity as well. 7Terbinafi ne is highly effective against dermatophyte infections and acts by blocking ergosterol synthesis. 10udies which compared the effectiveness of different antifungal agents in treatment of onychomycosis at the same time 7 are scarce.Moreover, treatment of OM is a problematic issue in clinical practice. 1,10ntos et al, 7 tried to study in vitro susceptibility of 52 isolates of Trichophyton rubrum and 40 of Trichophyton mentagrophytes to griseofulvin, terbinafi ne, itraconazole, ketoconazole, fl uconazole and cyclopiroxolamine.They used modifi ed NCCLS approved procedure M38-A.Finally, they concluded that terbinafi ne was the most effective in vitro against all isolates, followed by itraconazole, cyclopiroxolamine, ketoconazole and fl uconazole.
Conidial suspensions or mixed suspension of mycelial fragments and conidia growing in a rich medium are used in the NCCLS methodology for antifungal testing makes use of. 13,14Employing nail powder for antifungal testing, 10 mimics the course of a natural nail fungal infection, with antifungal treatment starting only after mycelial growth.
In this method the fungi must use the nails as their only nutrition by activation of keratinases secretion.In this in vitro model, using its natural substrate, the dermatophytes act with more similarity to in vivo clinical condition. 5,10,15,16herefore, response to antifungal agents which is normally reported as MIC reached form NCCLS models would reach more relevant and practical result.
Due to our fi ndings, the fi nal MIC which resulted after the incubation of dermatophytes beside nail powder was much more than the initial which was concluded from conventional MIC assay.In addition Osborne et al, 10 found that terbinafi ne Nail-MFCs obtained from in vitro method using nail powder were much higher than MFC values obtained after conventional assays.Also, they found that the cidal action of terbinafi ne was much slower than in conventional assays.Therefore the current model of culture may show the needs for high dose and long duration of antifungal treatment for OM.
On the other hand, by comparing the effi cacy of the assessed antifungal drugs, we found that terbinafi ne inhibited the growth of dermatophytes in the in vitro model more effectively.Osborne et al, concluded similarly. 10he higher activity of terbinafi ne in comparison to other tested antifungal drugs has been established during in vitro studies. 7,17Therefore our fi nding in this regard is not new rather than higher MIC which had been also achieved by Osborne et al. 10 In conclusion, the model described here offers a clinically mimicking condition for measuring the effi cacy of different antifungal drugs for the treatment of onychomycosis.Future studies on dermatophytes using other biomaterials such as hair powder as a part of culture medium may be useful for other clinical conditions.

Table 1 : Initial MIC* for different antifungal agents according to the type of dermatophytes
After incubation with nail powder we found that fi nal MICs statistically increased in comparison with initial MICs (Initial MIC: 19.42 ± 17.21 μg/ml, fi nal MIC 36.72 ± 33.77 μg/ml, paired t-test, P value = 0.0001) [Table