SOURCE:

Faculty: Health Sciences And Technology
Department: Medical Laboratory Science

CONTRIBUTORS:

Nnadi, N. E.
Enweani, I.B

ABSTRACT:

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeasts that are able to cause fatal neurological infections in both human and other mammals. Cryptococcosis is the most common fungal infection of the central nervous system and has a huge burden in sub-Saharan Africa. This study was aimed at characterizing this yeast, to determine its susceptibility against commonly used antifungals and extracts of some plants used in traditional medicine against mycotic infections and the role of intra-varietal mating in driving evolution within the VNII mating group, and to characterize Chorio-allantoic membrane(CAM) model for C. gattii. In this study, one hundred samples of pigeon droppings were collected in Jos, Plateau State, Nigeria. C. neoformans was isolated from three samples and initially identified using standard phenotypic and biochemical tests. The susceptibility of the isolates was tested using commonly used antifungals (fluconazole, itraconazole, voriconazole and flucytosine). To assess the effect of some plant extracts against Cryptococcus a total of three different plants were collected after oral interview with traditional medicine vendors in the various communities in Plateau State Nigeria and Nnewi in Anambra State, Nigeria. In vitro activities of acetone, methanol, hexane and dichloromethane extracts of three plants species (Swetnia mahogany, Cassia alata and Rauvolfia vormitoria) were determined using broth microbroth dilution assay method against Cryptococcus gattii. Each extract was incubated for 48hours and minimum inhibitory concentration of each extract was measured after 24hours and 48hours. Molecular analysis revealed that all three isolates belonged to C. neoformans genotype VNII, mating type α and were assigned to the sequence type ST43 by multilocus sequence typing analysis. ST43 was observed to belong to a separate cluster within the VNII clade. The isolates were also sensitive to all the antifungal drugs tested. MIC values were 0.03-0.12µg/ml for itraconazole, <0.03 µg/ml for voriconazole, 2-4 µg/ml for fluconazole, and 0.25 µg/ml for flucytosine. The control strain MICs were within the recommended ranges. Plants with the best activity were selected based on the lowest MIC, presence of clear zone on bioautograms indicating fungal activity and high total activity against C. gattii. The Acetone extracts of C. alata had the best activity with an MIC value of 0.16mg/ml after 24hrs which changed to 0.32mg/ml after 48hours and had a total activity of 1042ml/g and 500ml/g respectively. Another plant that had a good activity was the dichloromethane extract of the root of R. vormitoria with an MIC value of 0.032mg/ml both at 24hours and 48hours and total activity of 354ml/g. Both plants gave clear zones of inhibition on direct bioautogram. Phytochemical assay of the plants showed the presence of Alkaloids and flavonoids At a concentration of 107 cfu/ml,. two strains of C .gattii R272 and R265 showed similar virulence, and virulence higher than EJB18 and Malassezia sympodialis strain. The formation of lesions by the organism and re-isolation of organism from CAM suggests that the model can be used for evaluating the virulence of C.gattii. Comparison between C. neoformans environmental strain EN326 and clinical strain KN99a using the Gallaria monella model showed a low virulence for environmental strain. Studies on intra-varietal mating between EN28(MATα) and KN99a(MATa) yielded a wide range of phenotypes and genotypes that vary from both parents. Two diploids were isolated (P033 and P188), with haploids that showed increased fitness when exposed to harsh environmental conditions. In conclusion, the study reports the first molecular typing of environmental isolates of C. neoformans in Nigeria, the optimisation of cowitch seed agar as an alternative for Cryptococcus isolation,the establishment of CAM model as an alternative model for virulence determination for C. gattii and the establishment of CAM model as a medium for induction of hyphae in M. sympodialis. As an alternative therapy R. vormitoria and C. alata are potential antifungal sources against Cryptococcus