1. Mycological Study of Dermatomycosis in Population of Cairo, Egypt Salama A. Ouf 1, Tarek A. Moussa 1, Samar M. R. Eltahlawi 2, Alshimaa S.M. Abd- elmegeed 1 1 Botany and Microbiology Department, Faculty of Science, 2 Dermatology Department, Faculty of Medicine 2, Cairo University, Giza 12613, Egypt

2. Introduction Dermatophytes are a group of closely related keratinophilic fungi that infect keratihized tissues such as hair, nails and skin. Virtually, there is no human population free from these mycotic diseases. Dermatophytes have increased over the past few decades particularly in immune- depressed patients. Although the research regarding the synthesis of new antifungal drugs has intensified, however the treatment with systemic antifungal chemical agents such as ketoconazole, fluonazole and itraconazole derivatives has side effects, in particular, when these chemicals are used in long-term application. This has open the way for finding out an alternative to chemical drugs. One of the possible approach is to use ozone therapy which has powerful anti- microbial properties against viruses, bacteria, parasites and fungi due to oxidation reaction which occurs upon any collision between an ozone molecule and a molecule of an oxidizeable cellular components, particularly those containing double bonds, sulfhydryl groups, and phenolic rings.

3. Aim of study 1- The aim of this investigation was to study the occurrence and etiological agents of dermatomycosis and candidiasis among 640 patients attending dermatology clinics at three hospitals in Cairo, Egypt 2- Parallel study was undertaken to determine the prevalence of dermatophytosis among the patients who were suffering from diabetes, cancer and heart diseases. This study included 620 patients attending National Endocrine and Diabetes Institute, 280 at National Cancer Institute and 210 at National Heart Institute. 2- To investigate the effect of ozone on growth, cellular leakage and keratinolytic activity of the recovered dermatophytes

4. Total Total male / Total female Age groups (yr) Clinical types of tinea 61 - 7051 - 6041 - 5031 - 4021 - 3011 - 200 - 10 183 120 ----- 2298male Tinea capitis 63 ------ female 95 44 -- 6 - 2828male Tinea corporis 51 - 46144320female 44 211222824male Tinea cruris 23 -- 85523female 135 4555810854male Tinea pedis 90116322912 -- female 49 13 - 1462 -- male Tinea unguium 36 - 21614211female 74 41 --- 51620 - male Tinea versicolor 33 --- 11796female 60 26------ male Candidiosis 34 ----- 133female 640 310/33073082966673286 Total Table 1. Survey of patients suffering from skin mycosis due to dermatophytes and other fungi in three hospitals at Cairo classified according to age group

5. Table 2. Survey of patients suffering from dermatophytic and non-dermatophytic infections in three hospitals, classified according to clinical types and etiological agents Culture results of different ringworms Positive cultures Negative cultures Total Dermatophytes Non - dermatophytes Total Non- filamentousFilamentous Microsporum gypseum Trichophyton interdigitale Microsporum canis Trichophyton tonsurans Trichophyton mentagrophytes Trichophyton rubrum Malassezia furfur Unicellular ( Yeast cells) Aspergillus spp Fusarium spp Scopulariopsis spp Male 97436922132780604280 47 Female 51742502726 1011126313 Total 1424851194939531811721059347 640

6. Figure 1. Occurrence of different clinical types of tinea in 81 from total 620 patients suffering from diabetes Infected cases (%)

7. Figure 2. Occurrence of different clinical types of tinea in 82 from total 280 patients suffering from cancer Infected cases (%)

8. Figure 3. Occurrence of different clinical types of tinea in 30 from total 210 patients suffering from heart diseases Infected cases (%)

9. Table 3. Minimum inhibitory concentration (MIC) of ozone applied as a gas for 2 hours or as ozonized oil for 2 minutes against growth and spore germination of the test dermatophyte fungi. MIC (µg/ml)MIC a (µg/ml) Dermatophyte fungiDermatophyte fungi Ozonized oilOzonized oil b Gaseous ozoneGaseous ozone Spore germinationSpore germinationgrowth Spore germination growth 0.250.544Microsporum gypseumMicrosporum gypseum 0.250.544M. canisM. canis 1.02.0816Trichophyton interdigitaleTrichophyton interdigitale 1.02.0816T. mentagrophytesT. mentagrophytes 0.251.088T. rubrumT. rubrum a The minimum inhibitory concentration (MIC) is defined as the lowest ozone concentration that reduced growth or spore germination by 80% in comparison to the ozone-free controls. b Oil was dissolved in dimethyl sulfoxide (DEMSO).

10. Table 4. Effect of the minimum inhibitory concentration (MIC) of ozonized oil (for growth) on sporulation (log CFU/ml) of the test dermatophyte fungi Dermatophyte fungi Sporulation (log CFU/ml) % reduction a ControlOzonized oil ** Microsporum gypseum6.79 ± 0.324.90 ± 0.2798.71 M. canis6.79 ± 0.215.26 ± 0.3297.05 Trichophyton interdigitale5.65 ± 0.124.92 ± 0.2681.33 T. mentagrophytes5.61 ± 0.174.75 ± 0.3286.34 T. rubrum5.83 ± 0.085.28 ± 0.1172.06 a Reduction in sporulation calculated as percentage from the control value

11. Table 5. Change in conductance of the bathing solutions containing mycelia of some dermatophyte fungi previously treated with the minimum inhibitory concentration (MIC) of ozonized oil. Dermatophyte fungi Conductivity after 8 hrs (µmohs/g fresh weight) Leakage as % of total conductance ControlOzonized oil Total conductance a Microsporum gypseum24.00 ± 2.4144.03 ± 3.8249.44 ± 2.9840.51 M. canis24.64 ± 3.9841.90 ± 4.8749.50 ± 6.3234.87 Trichophyton interdigitale22.98 ± 3.0232.51 ± 4.0146.22 ± 4.9320.62 T. mentagrophytes21.51 ± 2.2131.03 ± 3.1844.97 ± 5.3221.17 T. rubrum22.92 ± 3.8828.02 ± 2.9244.29 ± 3.9811.52 a Total conductance was measured by adding 1ml chloroform.

12. Table10. Sugar amount in bathing solutions after 8 hours incubation of mycelia of some dermatophyte fungi previously treated with the minimum inhibitory concentration (MIC) of ozonized oil. Dermatophyte fungi Sugar amount in bathing solution (µg/ml) % increase in sugar permeability ControlOzonized oil Microsporum gypseum154 ± 18312 ± 14102.6 M. canis133 ± 09243 ± 1582.7 Trichophyton interdigitale108 ± 11159 ± 1147.2 T. mentagrophytes110± 10171 ± 1055.5 T. rubrum95 ± 08131 ± 1037.9

13. Table 11. Effect of the minimum inhibitory concentration (MIC) of ozonized oil on the activity of keratinase of some selected dermatophyte fungi. Dermatophyte fungi Keratinase activity (U/ml) % reduction ControlOzonized oil ** Microsporum gypseum8.21±3.861.05±0.9887.21 M. canis26.32±3.866.11±3.3376.79 Trichophyton interdigitale9.14±2.064.43±3.0451.53 T. mentagrophytes18.98±4.506.17±2.7067.49 T. rubrum22.00±4.7011.08±5.7449.64

14. Table 12. Effect of ozonized oil and terbinafine applied at 2 µg/ml on recovery of guinea pigs a artificially inoculated with some dermatophytes. Dermatophyte fungi Recovery (%) b Control Ozonized oil (2 µg/ml) c Terbinafine as reference drug (2 µg/ml) Microsporum gypseum0.00100 M. canis0.0085.8±6.192.9±4.9 Trichophyton interdigitale0.0087.5±3.683.3±4.8 T. Mentagrophytes0.0082.5±2.287.5±6.3 T. rubrum0.0079.7±5.079.7±4.1 a Thirty two guinea pigs were employed for this experiment. b Guinea pigs were examined after five weeks post inoculation. C Ozonized oil was applied once a day.

15. Conclusion Tinea capitis was the most common clinical disease (28.6%) in the clinic attending patients, followed by tinea pedis (21.1%) and tinea corporis (14.8%). Tinea cruris and tinea unguium were the least in occurrence (6.9 and 7.7%, respectively). Tinea versicolor was detected in 11.6%. The skin diseases due to Candida spp. were reported in 9.4% of skin mycosis. The most susceptible persons were children below 10 years (44.7%) followed by those aging 31-40 years (15%). Unicellular yeast was the most common etiological agent and Trichophyton tonsurans was the second most frequent causative agent accounting for 18.6%, followed by Microsporum canis (13.3%). Diabetes looks a risk factor for tinea pedis (6.61% of diabetic patients) and tinea corporis (3.23%). The effect of ozone in the form of ozonized oil on the sporulation, mycelia leakage of sugar and nutrients as well as activity of keratinase of the pathogens is apparently more efficacious than gaseous ozone. Generally, M. gypseum and M. canis were the most susceptible fungi while T. interdigitale and T. mentagrophytes were relatively resistant. On a trail to apply a control measure, the percentage recovery of M. gypseum- infected guinea pigs reached 100% in on application of 2 µg/ml ozonized oil. This result is equivalent to that obtained by using 2 µg/ml terbinafine as antifungal reference drug.

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