Department of Biochemistry, Cell and Molecular Biology
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Item Detection and Characterization of Epstein Barr Virus in Nasopharyngeal Cancers in Ghana(University of Ghana, 2019-07) Ayee, R.Purpose: Nasopharyngeal cancer (NPC) is a malignant tumor which is commonly associated with Epstein Barr virus (EBV). Two genotypes of EBV, genotype 1 and genotype 2, which are geographically restricted, are implicated in the pathogenesis of NPC. This study aimed at detecting and characterizing EBV genotypes involved in the pathogenesis of NPC in Ghana. Experimental design: Whole blood and biopsy samples were collected from 55 patients diagnosed of NPC by CT scan and endoscopy at the Ear, Nose and Throat Unit (ENT) of the Korle-Bu Teaching Hospital (KBTH). As control subjects, whole blood was collected from 53 individuals confirmed NPC negative or without known oncological diseases by CT scan and endoscopy. Association of NPC with risk factors such as gender, alcohol intake, smoking and consumption of salted fish, was evaluated. EBV was detected by amplification of Epstein Barr nuclear antigen 1 (EBNA-1) and genotyping by amplification of Epstein Barr nuclear antigen 2 (EBNA-2) using primers specific to genotypes 1 or 2. Viral load in blood and biopsy specimen was quantified using real-time polymerase chain reaction (PCR). Results: Risk factors such as gender, consumption of alcohol and smoking were not significantly (p > 0.05) associated with NPC development. There was a significant association (p< 0.05) of consumption of salted fish with NPC development. The frequencies of EBV positivity were 67%, 67% and 92% in NPC blood, NPC biopsies, and control blood samples, respectively. The predominant EBV genotype in blood specimen from cases was EBV genotype 2 (52%) and that of the control group was type 1 (62%). Statistically significant difference (χ2= 72.26, p = 0.001) was observed for the frequency of EBV genotypes in the NPC blood and controls. In biopsy specimen, the predominant EBV genotype was genotype 1 (58%). Median viral load (9×107 copies/ mL) in whole blood of NPC case subjects was significantly higher than median viral load (6×104 copies/ mL) in the whole blood of control subjects (p=0.001). Significantly higher (p < 0.001) median EBV DNA load (9×107 copies/ mL) was observed in NPC whole blood samples compared to that of NPC tumor biopsies (1.58×105 copies/mL). The median viral load of case samples infected with EBV genotype 1 was significantly higher (p = 0.001) than control samples infected with the same EBV genotype (genotype 1 median viral load in cases, 2 ×107 copies/ mL verses 29 ×103 copies/ mL genotype 1 median viral load in controls). Significantly higher (p = 0.001) median viral load was observed in cases infected with EBV genotype 2 when compared to control samples infected with the same EBV genotype (1×108 copies/ mL compared to 18 ×103 copies/ mL in cases and controls, respectively). Conclusions: In summary, gender, consumption of alcohol and smoking were not associated with NPC development in the current study whereas association was established between NPC and salted fish consumption. The frequency of EBV was higher in control subjects than NPC patients, confirming reports suggesting large number of the world’s population being asymptomatic carriers. Gender was not a risk factor of EBV infection in this study. This study identified EBV genotype 2 infection as the virulent genotype in Ghana and a possible risk factor in the development of NPC in Ghanaian patients. EBV genotype 1 was predominant in the control group and consistent with the genotype being relatively less virulent. Detection and quantification of EBV load can be used as a non-invasive biomarker for diagnosis of NPC.Item Isolation and Characterization of Antifungal Agents Produced by Wood Decaying Fungi From Ghana(University of Ghana, 2014-07) Adadey, S.M.Human systemic fungal infections have increased in the past decade due to the dramatic increase in immunocompromised patients hence the emergence of resistant fungal strains. It is therefore necessary to discover new antifungal compounds with novel mode of action. This study was aimed at isolating and characterizing antifungal agents from wood decaying fungi (WDF). The growth conditions for maximum production of bioactive agents from WDF were first tested. It was observed that mineral supplements from soil extract supported the growth and metabolite production of WDF. Media richness, culture volume and mineral supplementation were identified as the critical factors that influence the batch to batch consistency of metabolite production in WDF. Collections of 189 WDF were partially screened for their antifungal activity against Candida albicans. They were further screened against Saccharomyces cerevisiae in this project. Based on this screening, 33 of WDF were selected for mycelium isolation. The mycelia of 31 out of the 33 were successfully isolated on agar plates and stored called Plate Mycelium (PM). This served as a continuous fungal inoculum source. The 31 isolated WDF mycelia were then grown in broth and their metabolites were extracted with ethyl acetate. The antifungal activity of each WDF extracts was validated through a phenotypic assay screening using media conditions and mutant S. cerevisaie strains. In the chemical phenotypic array, the sensitivity of the C. albicans and S. cerevisiae cells to the WDF extracts was altered by chemical modifications in the YPD agar plates. This was used as a read out for validating the antifungal activities of the extracts. From the phenotypic screening, the best 6 WDF candidates with highly potent antifungal activity were selected. The mode of action of this top 6 WDF was examined by the antifungal activity pattern displayed against the mutant S. cereviaise cell.Item Isolation And Characterization Of Compounds From Wood Decay Fungi (Wdf) With Activity Against Gram- Positive Bacteria(University of Ghana, 2015-07) Akraifie, G.BFor decades, fungi have been a source of anti-infective substances, especially after the discovery of penicillin. However, in recent years, the efforts of pharmaceutical companies have dwindled due to the redundancy in the discovery of anti-infective compounds. Antibiotic resistance is also on the increase worldwide, and this poses a serious clinical threat. This study explored metabolites from indigenous Ghanaian Wood Decay Fungi (WDF) by screening a library of 180 WDF. Extracts obtained from these fungi were tested against Gram-positive organisms, Methicillin- sensitive Staphylococcus aureus (MSSA) and Methicillin-Resistant Staphylococcus aureus (MRSA). Out of 180 WDF screened, 48% had antibacterial activity against both susceptible and resistant strains of S. aureus, with 23.45% showing selective activity against only the resistant strains of S. aureus. Techniques to optimize the fermentation of WDF were explored by varying the closing mechanism of fermentation vessels using filter paper capping, foam capping, loose capping and tight capping. Filter paper-covered vessels were the most suitable for the production of extracts with consistent activity. Six different media were tested for maximum production of bioactive compounds. Yeast, Peptone, Malt extract, and Dextrose (YPMD) broth aided the production of consistent antibacterial activity among the six media tested. Inhibitors of histone deacetylases (iHDACs) were used to modify broth in which two fungi were cultured. iHDACs increased the antibacterial activity of the two fungal extracts. Using a phenotypic array assay that employed synergy between standard antibiotics and phenotype modulating compounds, fungal isolates taken through activity-guided fractionation revealed five potential compounds with antibacterial activity. This study is a proof of concept that Wood Decay Fungi (WDF) have the potential of producing secondary metabolites with anti-Gram positive activity. The study also established an assay that provides a predictive power of WDF with potential novel antibacterial compounds, as well as techniques for optimizing fermentation of WDF to produce ant ibacterial compounds.