Urea is commonly used as a keratolytic substance in the treatment of onychomycoses to improve the penetration of antifungal drugs in the lesion sites. The aim of the present study was to investigate the inhibitory action of urea on samples of dermatophytes in vitro.
METHODMinimum inhibitory concentration of urea was determined for 31 samples of dermatophytes cultured in Sabouraud-dextrose broth containing different concentrations (7.5% up to 40%) of urea. Absence of growth was the criterion adopted to determine the minimum inhibitory concentration.
RESULTSThe majority of samples (87%) were sensitive to urea at 12.5%, or less. 2 isolates of Trichophyton tonsurans and 2 of Trichophyton rubrum required 30%, and 40% urea, respectively, to be completely inhibited.
CONCLUSIONIn vitro results demonstrate inhibitory activity of urea on dermatophytes, suggesting that it could be used as an adjuvant in topical treatments.
A uréia é comumente usada como substância queratolítica no tratamento das onicomicoses no intuito de melhorar a penetração das drogas antifúngicas. O objetivo deste estudo foi investigar a ação inibitória in vitro da uréia em amostras de dermatófitos
MÉTODOSA concentração inibitória mínima da uréia foi determinada para trinta e uma amostras de dermatófitos semeadas em meio de cultura Sabouraud-dextrose contendo diferentes concentrações (7,5% até 40%) de uréia. Ausência de crescimento foi o critério adotado para a determinação da concentração inibitória mínima.
RESULTADOSA maioria das amostras (87%) foi sensível à uréia em concentrações de 12,5% ou menos. Apenas dois isolados de Trichophyton tonsurans e dois de Trichophyton rubrum foram inibidos completamente na presença de 30% e 40% de uréia, respectivamente.
CONCLUSÃOOs resultados in vitro demonstraram atividade inibitória da uréia sobre os dermatófitos, sugerindo que possa ser usada como um adjuvante em tratamentos tópicos.
Dermatophytes are fungi capable of invading keratinized regions such as skin, hair, and nails of human beings and animals, causing diseases known as dermatophytoses.1 Among the species of anthropophilic dermatophytes, Trichophyton rubrum is the most adapted to human beings and is one of the most important agents of tinea unguium, tinea pedis, tinea manuum, tinea corporis, and tinea capitis.2–10 Treatment with topical antimycotics are generally not indicated for hyperkeratotic type tinea pedis and onychomycoses. In these cases, griseofulvine and, more recently, itraconazole and terbinafine have been frequently administered. However, such drugs can cause intestinal and hepatic disturbances or interactions with other consumed drugs, precluding oral treatment.11
Several agents for topical use are commercially available for the treatment of superficial mycoses. The medication is prescribed according to the etiology of the fungus, site, and extent of the lesions. Clinical aspects of the host are important for establishing the choice of a topical or systemic treatment.12
Treatment is usually prolonged for hyperkeratotic lesions of the feet and nails. In order to shorten it, drugs associated with keratolytic agents have been tested. Studies have shown that bifonazole plus urea can be useful in this situation.12–14,16 The application of urea along with butenafine can also result in earlier improvement of dermatological symptoms of hyperkeratotic-type tinea pedis15 as well as when associated with lanoconazole.11
In summary, these studies have shown that the keratolytic/exfoliative action of urea can reduce the period of treatment of hyperkeratotic lesions. However, there are no reports on the inhibitory activity of urea on dermatophytes, except for the work of Faergemann & Swanbeck17 who investigated the in vitro action of urea against a sample of T. rubrum, demonstrating that the minimal inhibitory concentration (MIC) of a solution containing urea, lactic acid, and propylene glycol was lower in comparison to the same solution without urea.
The aim of this study was to evaluate in vitro the inhibitory activity of urea on dermatophytes samples.
MATERIALS AND METHODSMicroorganismsThirty-one samples of dermatophytes isolated from patients presenting skin (n = 18) and nail lesions (n = 13) were analyzed (Table 1).
Dermatophyte identification of 31 clinical samples studied
| Samples | Reference number | Location | Origin |
|---|---|---|---|
| T. rubrum | 11 | Inguinal region | Laboratory of Mycology – IMT |
| T. rubrum | 16 | Foot | Laboratory of Mycology – IMT |
| T. rubrum | 22 | Foot | Dermatology Service–HCFMUSP |
| T. rubrum | 25 | Foot | Dermatology Service – HCFMUSP |
| T. rubrum | 29 | Foot | Proença Laboratory |
| T. rubrum | 56 | Foot | Dermatology Service – HCFMUSP |
| T. rubrum | 68 | Foot | Proença Laboratory |
| T. rubrum | 70 | Foot | Proença Laboratory |
| T. rubrum | 109 | Nail | Laboratory of Mycology – IMT |
| T. rubrum | 114 | Nail | Laboratory of Mycology – IMT |
| T. rubrum | 115 | Nail | Laboratory of Mycology - IMT |
| T. rubrum | 117 | Foot | Laboratory of Mycology – IMT |
| T. rubrum | 118 | Foot | Laboratory of Mycology – IMT |
| T. rubrum | 119 | Foot | Laboratory of Mycology – IMT |
| T. rubrum | 121 | Glutea region | Laboratory of Mycology – IMT |
| T. rubrum | 126 | Nail | Laboratory of Mycology – IMT |
| T. rubrum | 154 | Nail | Proença Laboratory |
| T. rubrum | 155 | Nail | Laboratory of Mycology – IMT |
| T. rubrum | 156 | Nail | Laboratory of Mycology – IMT |
| T. tonsurans | 04 | Scalp | Laboratory of Mycology – IMT |
| T. tonsurans | 06 | Nail | Dermatology Service –HCFMUSP |
| T. tonsurans | 09 | Foot | Laboratory of Mycology – IMT |
| T. tonsurans | 24 | Foot | Dermatology Service –HCFMUSP |
| T. tonsurans | 34 | Nail | Laboratory of Mycology – IMT |
| T. tonsurans | 125 | Nail | Proença Laboratory |
| T. mentagrophytes | 10 | Foot | Laboratory of Mycology – IMT |
| T. mentagrophytes | 33 | Nail | Proença Laboratory |
| T. mentagrophytes | 83 | Nail | Laboratory of Mycology – IMT |
| T. mentagrophytes | 153 | Foot | Laboratory of Mycology – IMT |
| M. canis | 142 | Nail | Proença Laboratory |
| M. gypseum | 110 | Foot | Brigadeiro Hospital |
Initially, a direct examination of the samples treated with KOH 20% was performed by optical microscopy. Then, the samples were cultured in tubes containing mycobiotic agar and maintained at 25°C. The macroscopic aspect of the colonies was observed during a 2-week period.
To identify the isolate at genus and species level, microcultures of the dermatophytes were carried out in potato agar maintained at 25°C, followed by microscopic examination of the slides stained with lactophenol cotton blue.
Susceptibility to ureaIn order to determine the in vitro sensitivity to urea, all the dermatophytes samples were cultured in 18 x 180 mm tubes with 10 mL of Sabouraud-dextrose broth having the following concentrations of urea: 7.5%, 10%, 12.5%, 20%, 30%, and 40%. All the cultures were incubated at 30°C. Tubes containing only Sabouraud-dextrose broth as culture medium were used as positive growth controls.
Minimal inhibitory concentration (MIC) determinationAbsence of growth was the criterion adopted to define the MIC of urea, which was determined between the 7th and 10th day, when the control tube had well developed typical colonies.
The growth of the samples during the incubation time was recorded and graded as follows: C = control tube (plentiful growth); (+) = weak growth; (++) = moderate growth; (+++) = plentiful growth; (-) = absence of growth (Figure 1).
RESULTS
The causative agents in 31 samples of dermatophytes from patients presenting skin and nails lesions were identified as Trichophyton rubrum (19), Trichophyton tonsurans (6), Trichophyton mentagrophytes (4), Microsporum gypseum (1), and Microsporum canis (1) (Table 1).
Of note is the high frequency of T. rubrum in feet and nails in the samples that showed growth inhibition atconcentrations lower or equal to 10%.
Two T. tonsurans isolates were sensitive at 30% urea, and 2 T. rubrum isolates were sensitive at 40% (Table 2).
Growth index of 31 isolates of dermatophytes sown in Sabouraud-dextrose broth at 30°C in the presence of different concentrations of urea
| Concentrations (%) | |||||||
|---|---|---|---|---|---|---|---|
| Urea Samples | C* | 7.5% | 10% | 12.5% | 20% | 30% | 40% |
| T. rubrum (11) | +++ ** | +++ | +++ | − | − | − | − |
| T. rubrum (16) | +++ | − | − | − | − | − | − |
| T. rubrum (22) | +++ | + | + | − | − | − | − |
| T. rubrum (25) | +++ | + | + | − | − | − | − |
| T. rubrum (29) | +++ | − | − | − | − | − | − |
| T. rubrum (56) | ++ | − | − | − | − | − | − |
| T. rubrum (68) | +++ | +++ | +++ | − | − | − | − |
| T. rubrum (70) | +++ | ++ | − | − | − | − | − |
| T. rubrum (109) | +++ | − | − | − | − | − | − |
| T. rubrum (114) | +++ | +++ | +++ | ++ | ++ | + | − |
| T. rubrum (115) | +++ | +++ | +++ | ++ | ++ | ++ | − |
| T. rubrum (117) | +++ | +++ | +++ | − | − | − | − |
| T. rubrum (118) | +++ | +++ | +++ | − | − | − | − |
| T. rubrum (119) | +++ | +++ | +++ | − | − | − | − |
| T. rubrum (121) | +++ | +++ | +++ | − | − | − | − |
| T. rubrum (126) | +++ | ++ | ++ | − | − | − | − |
| T. rubrum (154) | +++ | − | − | − | − | − | − |
| T. rubrum (155) | +++ | + | − | − | − | − | − |
| T. rubrum (156) | +++ | + | + | − | − | − | − |
| T. tonsurans (04) | +++ | + | + | − | − | − | − |
| T. tonsurans (06) | +++ | ++ | − | − | − | − | − |
| T. tonsurans (09) | +++ | +++ | ++ | ++ | ++ | − | − |
| T. tonsurans (24) | +++ | ++ | − | − | − | − | − |
| T. tonsurans (34) | +++ | +++ | +++ | − | − | − | − |
| T. tonsurans (125) | +++ | +++ | +++ | ++ | ++ | − | − |
| T. mentagrophyte(10) | +++ | +++ | +++ | − | − | − | − |
| T. mentagrophyte(33) | +++ | ++ | + | − | − | − | − |
| T.mentagrophytes(83) | +++ | ++ | − | − | − | − | − |
| T. mentagrophytes(153) | +++ | + | − | − | − | − | − |
| M. gypseum (110) | +++ | − | − | − | − | − | − |
| M. gypseum (161) | +++ | ++ | − | − | − | − | − |
| M. canis (142) | ++ | − | − | − | − | − | − |
The sample distribution according to the lesion site is presented in Table 3.
DISCUSSIONMost dermatologists use topical formulations of antifungal drugs. The choice of treatment depends on the etiology, location, clinical form, and extent of the fungal lesions. In many cases, the patient's underlying conditions determine the treatment choice: creams, solutions, powders, gels, or capsules.12
The therapeutic schemes for hyperkeratotic type tinea pedis and onychomycosis, mainly for the toenails, are seriously problematic, because the results with griseofulvine are not sufficiently satisfactory and side effects are frequent. More recently, itraconazole and terbinafine have been used. These antifungal agents produce fewer side effects, but their high cost can make the treatment impractical.
Additionally, topical antifungal drugs are also unsatisfactory due to the poor penetration into the nails. However, earlier studies have shown that the use of propyleneglycol associated with urea and lactic acid produces effective results for the treatment of the nail tissue. Among 23 patients presenting onychomycosis caused by Candida albicans and Trichophyton rubrum, 21 had good in vivo responses with only topical treatment using this combination.17
The association of topical antifungal drugs with urea has been shown to improve treatment outcomes. Association of bifonazole with 10% urea to treat patients with tinea pedis produced clinical improvement in 92% of the cases after 12 weeks. Additionally, the treatment of the hyperkeratotic-type tinea pedis with topic butenafine hydrochloride plus 20% urea provided an earlier dermatological improvement compared to the use of oral antifungal drugs.15 Regarding etiologies among the dermatophytoses, T. rubrum has been shown to have a high incidence as a causative agent.16,18,20 In our study, the most frequently isolated dermatophyte from different body regions was T. rubrum.
An earlier study evidenced the antimycotic activity of urea at 5% against a T rubrum isolate in vitro.17 Our data show that the majority of samples, including T. rubrum, were sensitive to 12.5% urea. Of note is that only 4 isolates, 2 of T. tonsurans and 2 of T. rubrum, respectively, required 30% or 40% urea for their complete inhibition. These data are related to clinical observations, because lesions caused by T. rubrum are sometimes difficult to treat, and recurrence frequently occurs.21
These in vitro results demonstrate the inhibitory activity of urea on dermatophytes, suggesting that this keratolytic agent may also have a fungicide action and that it could be used as an adjuvant in topical treatment. Further studies correlating time of treatment and cure percentage of patients using topical medications in the presence of urea must be conducted for a better evaluation of its antimycotic action.