SOURCE:

Faculty: Biosences
Department: Applied Microbiology And Brewing

CONTRIBUTORS:

Udemezue, O.I;
Oyeka, C.A;

ABSTRACT:

The increased risk of fungal diseases especially opportunistic fungal infections such as candidiasis, emerging fungal pathogens, limited range of antifungal drugs, high level of toxicity and development of resistance to the available antifungal drugs have increased the demand for more clinically effective and safer antifungal agents. The continuous search for more potent, cheaper and non-toxic raw materials to feed the pharmaceutical industries is inevitable. The aim of this work was to evaluate the therapeutic potentials of natural compounds (potash compounds and natural stones such as palm ash, potash alum, trona, blue stone, black stone and sulphur stone) for cutaneous candidiasis treatment using murine models. Four hundred and fifty high vaginal swab specimens were collected from patients suspected of having vulvo-vaginal candidiasis. These were inoculated on Sabouraud dextrose agar supplemented with 50µg/ml chloramphenicol and incubated aerobically at 25oC for 48hours. The yeast isolates were identified based on their morphological, physiological, biochemical and molecular characteristics. Antimicrobial activity of the natural compounds on the Candida isolates were evaluated using in vitro (agar-well diffusion) and in vivo methods (albino rat model). The Minimum inhibitory concentration (MIC) and Minimum fungicidal concentration (MFC) of the agents were comparatively determined using broth dilution and micro-dilution methods. Full blood count of the test animals before and after immunosuppression was analysed using hemoanalyser. The organic and inorganic chemical constituents of the natural compounds were determined using GC-MS and X-ray fluorescence technology respectively. The in vivo toxicity screening of the natural agents was done using Draize’s skin irritancy test, skin sensitization test and necropsy. The in vivo antimicrobial assay was done using eight-month old healthy albino rats weighing 220g – 240g. The animals were immunosuppressed intraperitoneally using 0.1ml cyclophosphamide (100mg/kg body weight). They were inoculated with a loopful of the isolates (equivalent to 1 x 108cfu/g) percutaneously and carefully observed for clinical signs and symptoms of candidiasis. Out of the 450 HVS specimens collected, 188 (42%) were positive for Candida growth. Candida albicans, 96 isolates (51%), C. tropicalis, 53 isolates (28%), C. glabrata, 28 isolates (15%) and C. parapsilosis, 11 isolates (6%). Among the potash compounds analysed, trona gave the highest inhibition zone diameter (IZD), 31.00±1.41mm against C. glabrata, potash alum, 30.00±1.41mm against C. parasilopsis and palm ash, 17.50±0.71mm against C. glabrata at 200mg/ml. Blue stone was the best among natural stones, 31.50±0.71mm against C. tropicalis, sulphur stone, 18.50±0.71mm against C. parapsilosis and Black stone, 0.00mm for all the isolates at 200mg/ml. The positive control ketoconazole (at 200mg/ml) gave the IZD of 21.00±1.41mm against C. albicans, 21.50±0.71mm against C. tropicalis, 20.50±0.71mm against C. glabrata and 19.00±1.41mm against C. parapsilosis. The MIC and MFC of the test agents varied among the Candida isolates. The MIC of blue stone for C. albicans was (50mg/ml), Potash alum (50mg/ml), Trona (100mg/ml), Sulphur stone (200mg/ml), Black stone (200mg/ml), Palm ash (100mg/ml) and Ketoconazole (50mg/ml). The combined activity of the agents (at 1:1 ratio) using their MIC and fractional inhibitory concentration (FIC) values against the isolates also varied. The agents exhibited synergism against C. albicans (FIC<1), indifference for C. tropicalis (FIC between 1 and 2) while antagonism for C. glabrata and C. parapsilosis (FIC>2). Palm ash + trona and palm ash + potash alum + trona combinations were antagonistic against all the isolates (FIC>2). During the in vivo studies, the signs and symptoms of cutaneous candidiasis disappeared gradually 5 – 7days during treatment with various formulations of the natural compounds. The immunosuppressed group developed infection 7 days after inoculation while the normal group developed infection 9 days after inoculation. There was a drastic decrease in the white blood cells count five days after the immunosuppression; Neutrophil count (53.1% to 25.4%), Monocyte (6.7% to 2.3%), Eosinophil (1.7% to 1.3%) and basophil (0.6% to 0.5%). Histological analysis of stained infected rat skin sections revealed intense peri-adnexal infiltration of inflammatory cells, inflammed sebaceous units and malformed collagen bundles. Tissue regeneration was much quicker in treated groups compared to the negative control groups. Ketoral cream (ketoconazole) was used as the positive control. Total yeast count was done at 3 days intervals during treatment to evaluate the effectiveness of the agents in reducing the yeast population in vivo. The yeast population declined gradually with time (from 108cfu/g to 102cfu/g), thus showing the in vivo effectiveness of the antimicrobial agents in treatment of cutaneous candidiasis. The major inorganic components of the natural compounds were Blue stone: Cu(43.7%), S(29.4%), As(0.0015%), Pb(0.002%), Ni(0.005%); potash alum: S(44.5%), Al(14.49), Pb(0.0009%), As(0%), Ni(0.0004%); Trona: Cl(5.81%), Si(22.6%), Pb(0%), As(0.003%), Ni(0.0007%); Sulphur stone: S(94.72%), Al(3.5%), As(0%), Pb(0.0006%), Ni(0.00008%); Black stone: S(1.6%), Cl(0.98%), Cu(0.234%), Pb(0.001%), As(0%), Ni(0.0021%) and Palm ash: K(28.84%), Cl(15.21%), S(3.36%), Urea(8.6%), Pb(0.00074%), As(0.00052%), Ni(0.0007%). The organic constituents are basically naturally occurring fatty acids including vaccenic, decanoic, oleic and linoelaidic acids. In vivo toxicity analysis of the natural compounds showed no observable histopathological signs as seen in the architecture of the collagen bundles, blood vessels and sebaceous units described in the stained skin sections. The findings revealed that potash compounds and natural stones are clinically safe and effective in treatment of cutaneous candidiasis and could serve as good alternatives to conventional antifungal antibiotics.