Keywords: Antifungal, Microdilution, dermatophytes, Children.

Dermatophytes are communicable fungal disease caused by three genera of fungi namely, Trichophyton, Microsporum and Epidermophyton. Several varieties of tropical and systematic preparation are reported to have reasonable activity in these diseases and are being used in treating these superficial mycotic infections (1,2). Although in the last few years, there has been a steady introduction of new drugs by either modification of pre-existing or entirely new chemical classes of antimycotics, there has also been an increased interest in clinically relevant susceptibility testing (3). This has become necessary due to a considerable increase in the incidence of dermotophytoses all over the world especially in underdeveloped countries such as Nigeria (4-7) among the elderly and in immunocompromised patients.(8). In developed countries of Europe and America and fast developing countries of Asia, data on the antifungal susceptibility of dermatophytes could not be said to be entirely scanty, the case appears different in African countries, such as Nigeria, where unfortunately, the occurrence of dermatophytosis is endemic. Expansion of information on invitro susceptibility testing of dermatophytes will provide data that will help in developing or selecting drug regimens (9). In addition, the various cases of imported dermatophyte infection across different countries and the variability in the antimycotic properties of similar strains obtained across different geographical locations of the world make exchange of information on susceptibility data across different countries necessary. We carried out susceptibility studies on clinical isolates obtained in children previously. We believe that our data will also help clinicians to choose appropriate antifungal agents for a successful treatment outcome in their patients especially in countries like ours where immediate laboratory diagnosis for this infections are not readily performed before treatments are administered.

Test isolates

Seventy-one isolates of dermatophytes were evaluated. Out of this 27 were Trichophyton tonsurans, 15 were Trichophyton soudanense,7 were Trichophyton violaceum, 5 were Trichophyton rubrum, 4 were Trichophyton verrucosum, 9 were Trichophyton mentagrophytes, 3 were Trichophyton schoenlenii and 2 each of Epidermophyton flocossum and Microsporum audounii. Three American typed culture collections (ATCC) quality control organisms were used: Candida parasilopsis ATCC22019, Trichophyton mentagrophytes (ATCC 40004) and Candida krusei ATCC 6258. These dermatophytes were mainly isolates from different body sites collected from children during previous investigational study. (4,5). The cultures were maintained on Saboraud dextrose agar slants supplemented with cycloheximide, chloramphenicol and genticin (SAB-CCG). Freshly growing cultures on SAB-CCG slants were transferred to oatmeal agar slants two weeks prior to the study to enhance conidial production as proposed previously (10)

Antifungal agents

Five antifungal agents were used, itraconazole (ITR) and ketoconazole (KET) (Jansen), fluconazole (FLU) (Pfizer), terbinafine (TER), (Novartis) and griseofulvin (GRI) (Schering plough). Fluconzole and ketoconazole were dissolved in sterile distilled water while the rest were dissolved in 100% dimethylsulphuroxide (Sigma-Aldrich). They are subsequently prepared as stock solutions and stored at appropriate temperature.

Invitro tests for susceptibility

Drug dilutions/medium: RPMI 1640 (Sigma- Aldrich) with L- glutamine but without sodium bicarbonate and buffered at PH 7 with MOPS was used for susceptibility testing.

Preparation of Inoculums

The method previously described by Norris et al (11) was used. Briefly; conidial suspensions of dermatophytes prepared from seven to fourteen day old culture grown on oatmeal cereal agar slants were made. Cultures producing between fifty and hundred conidia per field of view, for at least five different fields of view, were selected for testing. The selected slants were flooded with 0.85% sterile saline and swabbed with a cotton wool tip applicator. The resulting mixture of conidia and hyphal particles were subsequently transferred to a sterile tube. After settling for about 15-20 minutes, the upper layer (about 2ml) of the suspension was removed and adjusted for 80-85% transmission using a colorimeter. This corresponds to an inoculum of 1-5 x 106 CFU/ml. The inoculum was adjusted to 5ml with saline (0.85%) and diluted further in RPML 1640 to achieve a final concentration of 2-5 x 103 CFU/ml.

Testing procedure

Following CLSI guidelines M 27-P (12), Micro dilution plates were set up. Each test plate had two drug free growth controls, one with the media alone (growth control) and the other with media containing an equivalent amount of solvent used to dissolve the antifungal drug (solvent control). The plates (two for each) were incubated at 300c and 350c respectively and read visually after 4 and 5 days of incubation. Growth was normally checked after 48hrs post inoculation. Endpoint determinations were made by visually comparing the growth in the wells containing the drug with the growth in the solvent control well. MIC ranges were obtained for each species-drugs combination tested. To facilitate comparisons of the activities of the drugs, the MIC were reported as the concentration at which 50% (MIC50) and 90% (MIC90) of the isolates were inhibited.

The in vitro susceptibilities of 71 isolates of dermatophytes to terbinafine, itraconazole, ketoconazole, fluconazole and griseofulvin are summarized in Table 1. The data are presented as MIC ranges and, where appropriate, as the drug concentrations required to inhibit 50 and 90% of the isolates of each species (MIC90 and MIC50 respectively). The readings were obtained after 7 days of incubation. All the 71 isolates of dermatophytes tested were susceptible to the five antifungal drugs used in the study. The MIC of all the quality control strains used were within established ranges (data not shown) (13).

Although detectable growths were noticed after four days of incubation in some cases, majority of the isolates had pronounced growth after 5 days. We observed no major differences in incubating at 30 or 350C,but our results reflect readings recorded at 350C. However, no resistance was recorded among all the isolates. The isolates were less susceptible for griseofilvin and fluconazole with MICs ranging from 0.125 - 16.0 and 0.25- 64ug/ml. The MIC90 range for them respectively is between 2.0-8.0 ug/ml for griseofulvin and 4.0-16.0ug/ml for fluconazole. Terbinafine was the most effective drug against all isolates of dermatophytes since the MIC90 range was between 0.01-0.07mg/ml. The order of in vitro activity is therefore terbinafine > itraconazole > ketoconazole > griseofulvin > fluconazole

This is the first large -scale in vitro susceptibility testing of dermatophytes obtained from Nigeria against a wide range of commonly used antifungals in our country. Monitoring antimicrobial resistance is useful because apart from tracking and detection of resistance trends by microorganisms, it is also gives clues to emerging threats of new resistance. This serves among other things, in assessing interventional efforts and empirical treatments recommendations (14). In the past, several authors have performed in vitro susceptibility tests on various strains of dermatophytes (8, 10,11). However, there appears to be a lack of data on susceptibility studies of isolates from the West African sub region like Nigeria where this infection is endemic (4-5). We observed no major differences in the MIC endpoints by incubating at 300c or 350c. Information available in the literature from other authors (10, 15, 16) indicated that 4 days of incubation was sufficient to observe noticeable growth in the control wells. Our findings are also in agreement. We therefore recorded our MIC values after 4 days of incubation. However, studies from Santos and Hamden (17) and fromBelkys. Fernandez-Torres et al (18) are not in similar agreement. In another study by Fernadez-Torres and co-workers (19), 508 strains belonging to 24 species of dermatophytes were tested against conventional (itraconazole and fluconazole) and some newer antifungal agents like voriconazole and UR-9825. Our results on itraconazole and fluconazole are similar to those in their study. This tends to support the fact that incubating after 4 or 7 days does not have a significant impact on the MIC readings. Nevertheless, our result on terbinafine as the most active agent for example, agrees with the observation from previous authors in other continents and region (17,18). This antimycotic showed an excellent in vitro potency and broad-spectrum activity against all the tested species. This suggests that terbinafine can be used to treat a majority of dermatophytic infections especially those showing high MIC values on the azoles, such as fluconazole. Although we did not include the newer antifungals such as posaconzole, voriconazole etc in our study, we noticed in literature, their promising antifungal activities. In Nigeria, these antifungal are relatively new and not readily available, affordable and widely used as the ones we have tested. This buttresses our initial observation that most patients with dermatophytoses resort to use of some medicinal plants as a preferred treatment choice apparently due to inability to afford the orthodox drugs (4). It will be of interest to state that all isolates used in our study were obtained from patients not previously on any antifungal treatment. Interestingly; there was no record of resistance in our study even though some agents recorded high MIC values than others. From our data, ketoconazole appears to be next choice in terms of in vitro activity after terbinafine and itraconazole. This information among other things will assist clinicians to monitor trend and be able to choose effective medications for treating patients with dermatophytoses, especially in countries like Nigeria where dermatophytoses have become a public health problem and have remained endemic (5).