Invasion of the nail plate by a dermatophyte is referred to as tinea unguium in humans. There are two main types of nail involvement: invasive subungual (distal and proximal) and superficial white mycotic infection (leukonychia trichophytica). T. rubrum
and T. mentagrophytes
, respectively, are the most common dermatophytes of this infection [4
There are few reports of the animal model of tinea unguium. One study used guinea pigs [73
], and the other used rabbits [84
]. This section describes the two animal models.
4.1. Animal Model of Tinea Unguium in Guinea Pigs
Uchida et al. reported that T. mentagrophytes
could infect the nail when the tinea pedis model in guinea pigs would extend the duration of the experiment [73
]. Subsequently, the tinea unguium model, which was modified by the above model, was reported and evaluated drug efficacy [83
]. The method and the evaluation results of drug efficacy in tinea unguium using guinea pigs were as follows.
The arthrospores of T. mentagrophytes SM-110 were suspended, and the suspension was adjusted to give a concentration of 108 spores/mL. Two paper disks were immersed by the fungal suspension and applied between the toes of the hind paw (between the second third toes and between the third and fourth toes) with a foam pad, then fixed with adhesive elastic tape (day 0 after infection). The disks were removed on day 21 after infection. The antifungal treatment, topical KP-103, amorolfine, and terbinafine and oral terbinafine, started on day 60 after infection when the invasion of T. mentagrophytes SM-110 into the nail was confirmed; treatment continued for 30 consecutive days. The therapeutic efficacy of tinea unguium was evaluated by the culture method to avoid the drug carryover effects by using dialyzed samples that have been digested by enzymes. In the result, topical amorolfine and topical or oral terbinafine were ineffective even in terms of reducing the fungal burden. In contrast, topical KP-103 significantly reduced the fungal burden in the infected nails compared with the burdens found in the vehicle- and oral-terbinafine-treated groups.
This model was able to evaluate drug efficacy by two administration routes, oral and topical. Furthermore, this model can prevent drug carryover in the recovery culture and assess the pure viability of the fungi. However, this evaluation method is applicable only to water-soluble drugs. Thus, further consideration in the case of lipid-soluble drugs is necessary.
4.2. Animal Model of Tinea Unguium in Rabbits
We established an animal model using rabbits with confirmed fungi in the deep layer of the nail under an immunosuppressive condition [84
]. In this section, we describe the method and results of the drug efficacy of our model.
In some preliminary studies, three points were concluded: first, microconidia were more suitable than arthroconidia with regard to infection rate; second, the postinfection period from the end of infection to nail sampling was needed to maintain high infection rates; and third, administration of an immunosuppressant was essential to make T. mentagrophytes invade the nail to establish a reproducible infection. Subsequently, we performed experiments aimed at setting the suitable postinfection period, and the protocol for this experiment was as follows.
The nails of rabbits were immunosuppressed with injections of methylprednisolone acetate intramuscularly prior to application of 0.2
mL of fungal suspension (108
microconidia/mL) of T. mentagrophytes
TIMM2789 at a site between the lunula and the proximal nail fold. The nail plates of the first-to-third toes of the hind paw were wrapped together with a gauze patch and finger cot, and 0.5
mL of sterile water was injected into the finger cot to produce a culture environment around the nail that was seemed suitable for fungal growth. This condition was maintained for the duration of the infection for 2 weeks with no other intervention. The finger cot and the gauze patch were removed after 2 weeks of exposure, and this condition was maintained during for 0, 2, or 6 weeks without finger cot and gauze patch; this was termed the postinfection period. After each postinfection period was completed, the animals were sacrificed, and the nails were removed from the paw and treated histopathologically.
In the results, some of the infected nails became cloudy on gross appearance, which was similar to the findings with human onychomycosis. With a longer postinfection period, these findings were fully confirmed. On histopathological examination, hyphae of T. mentagrophytes penetrated the nail plate, and some invading fungi reached the nail bed. The infection rate in the sample at 0, 2, and 6 weeks after infection was 57%, 87%, and 93%, respectively. In addition, fungi proliferated and moved distally into the nail plate depending on the duration of infection. The presence of subungual abscess with associated necrosis of the epithelium of the nail bed or matrix was confirmed near the fungi in the nail plate. Above all, a high infection rate was obtained by 2 weeks inoculation with microconidia of T. mentagrophytes TIMM2789 and postinfection periods of more than 2 weeks were required.
Subsequently, the experiment for drug efficacy was confirmed for the topical antifungal agents, 8% ciclopirox nail lacquer and 5% amorolfine nail lacquer, using this model. The therapeutic period was set as 4 weeks after a 2-week infection period. The animals in the untreated control group underwent the process of infection and removal of the material used for this process, but they were not exposed to the test agents. The next day after the last treatment, the animals were sacrificed and the nails were removed from the paw for histopathological and microbiological examinations. In the microbiological examination, the infected nail intended for evaluation using culture recovery was cut into 10 pieces in cross-sections, and each nail piece was cultured on Sabouraud dextrose agar for 2 weeks at 28°C. A nail piece that had confirmed fungal growth was assessed as culture positive, and a nail with more than one culture-positive piece was considered fungus positive.
In the results of microbiological examination, a statistically significantly lower rate of culture positivity was found in the 5% amorolfine nail lacquer group for comparing the infection rate to that in both the control and 8% ciclopirox nail lacquer groups. Additionally, when the rate of culture positivity in the drug-treated groups was subtracted from that in the nontreated group, the differences were 54.2% on 5% amorolfine nail lacquer and 8.3% on 8% ciclopirox nail lacquer. This figure was similar in the clinical reports [85
This is the first report of fungal behavior in the nail plate in an experimental animal model of onychomycosis. Our experimental animal model succeeded in encouraging T. mentagrophytes to invade the deeper layers of the nail plate. The findings in our model were similar to the clinical diagnoses of the proximal subungual type (PSO). Furthermore, the efficacy of this model was close to the clinical cure rate. Further research using this model may be able to clarify the pathogenesis of onychomycosis and contribute to the development of drugs that match the clinical efficacy.