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Item Pathogenomics And Antimicrobial Resistance Analysis In Neisseria Gonorrhoeae(University Of Ghana, 2021-01) Agbodzi, B.Gonorrhoea is a poorly controlled public health problem. With the global emergence of resistance to first line antibiotic treatment options, the infection has been predicted to be untreatable in the near future. This emerging trend highlights the need for constant genetic surveillance to unravel the mechanisms of resistance and inform therapy. This study therefore, sought to perform whole genome characterization of N. gonorrhoeae collected in Ghana to identify lineages of circulating strains, their antimicrobial resistance (AMR) and some virulence determinants. Gonococci isolates were cultured on gonococcal (GC) medium and identified using the API NH kit (Biomerieux, France). Genomic DNA was extracted from N. gonorrhoeae isolates using the QIAamp® DNeasy Ultraclean Microbial kit (Qiagen, Hilden, Germany). Whole genome sequencing (WGS) was performed on 56 isolates using both the Oxford Nanopore MinION and Illumina MiSeq sequencing platforms. The Comprehensive Antimicrobial Resistance Database (CARD) and PubMLST Neisseria database were used to catalogue chromosomal and plasmid genes implicated in AMR and assign sequence types (STs). The core genome MLST (cgMLST) approach was used for comparative genomics. The Virulence Factors of Pathogenic Bacteria Database (VFDB) was used to annotate virulence factors. In vitro resistance measured by disc diffusion revealed that (56)100%, (51)91% and (50)89.3% of the isolates were resistant to tetracycline, penicillin and ciprofloxacin respectively, while for the E-test method, (54)96.4%, (51)91% and (49)87.5% respectively were recorded. Four isolates exhibited reduced susceptibility to both cefixime and ceftriaxone as measured by disc diffusion. For these isolates, MIC ranges of 0.004 – 0.016 μg/ml and 0.016 - 0.75 μg/ml for ceftriaxone and cefixime respectively were recorded. No spectinomycin and azithromycin resistance was recorded using the E-test method. A total of 22 STs were identified by Multi-Locus Sequence Typing (MLST), with ST-14422 (n=10), ST-1927 (n=8) and ST-11210 (n=7) being the most prevalent. Six novel STs were also identified and submitted for the assignment of new sequence types (ST-15634-115641). Seven clusters of isolates with distinct AMR genotypes were identified after the cgMLST analysis, highlighting the presence of genome wide genetic variation. All isolates harboured chromosomal AMR determinants that confer resistance to beta-lactam antimicrobials and tetracycline. A total of (49)87.5% and (13)23% isolates contained fluoroquinolone and macrolide resistance markers respectively. Plasmids were highly prevalent: pTetM and pBlaTEM were found in 96%, and 92% of isolates, respectively. All isolates possessed the PI (B) variant of the porB gene which is associated with localized infection while high antigenic variations in the pillin genes was also detected. The study highlighted the need for constant genomic surveillance with the looming possible emergence of cephalosporin resistant isolates and isolates with highly variable antigens which could severely impact disease treatment.Item In Vitro Studies Of The Effect Of Anopheles Gambiae Midgut Bacteria On The Development Of Plasmodium Falciparum(University Of Ghana, 2021-12) Ametsi, Williams GodwinDuring blood feeding, female Anopheles mosquitoes may ingest Plasmodium gametocytes which undergo transformation in the gut and develop into sporozoites that are infectious to humans. Bacteria inhabit the mosquito gut, and the number and diversity of these bacteria change following blood feeding. The presence of some bacteria species results in the reduced intensity of developing Plasmodium parasites. Little attention has been given to understanding this direct mechanism of bacteria on Plasmodium parasites, and the effects of bacteria on malaria parasite developmental genes are not completely understood. This limits the scope of how gut bacteria, for example Enterobacter and Serratia, which have been found with anti-Plasmodium effects can be further explored for alternative disease control strategies. Therefore, this study investigated the lethal effect of cell-free secreted bio-products of E. cloacae and S. marcescens on a key Plasmodium parasite developmental gene (Gamete release gene, GAMER) for its potential as a target for malaria transmission-blocking. Plasmodium falciparum 3D7 and Dd2 cultures at 1% parasitaemia were independently exposed to spent Luria-Bertani (LB) medium from varying concentrations of Enterobacter cloacae and Serratia marcescens. The parasite killing effect of the bacteria were assessed with SYBR green fluorescent assay after 48 hours of co-culture. Spent media with final bacteria concentration between 10e+10-10e+20 reduced parasitaemia (P<0.001) compared to parasite culture without bacteria treatment. Using real-time (quantitative) PCR, it was found that the expression of GAMER was down regulated by 2 folds after 1 hour of screening P. falciparum 3D7 with cell-free spent medium of E. cloacae cultured for 8 hours in LB broth (Ec-8). However, the expression of GAMER was unaffected after 6 and 12 hours of screening P. falciparum 3D7 with Ec-8. These data provide information for further studies on gene and protein targets for transmission blocking interventions.Item Selection Dynamics Of Circumsporozoite Protein (Csp) Vaccine Target In Ghana: The Contribution Of Human Leukocyte Antigen (Hla) Variation(University Of Ghana, 2021-07) Kpaka, J.N.Implementation of RTS,S/AS01 vaccine for malaria is underway in three (3) African countries, Ghana, Kenya, and Malawi. This vaccine, which targets the Plasmodium falciparum circumsporozoite protein (CSP) provides partial protection for infants and children against clinical and severe malaria infections. Reasons for this reduced efficacy or immunogenicity are poorly understood, but CSP variation has been implicated. Human leukocyte antigen (HLA) has also been observed to influence RTS,S-mediated protection. This study aims to define the variants of CSP and determine its distribution between Begoro and Cape Coast in Ghana over three years. Further, the influence of HLA genotype in terms of parasite frequency and RTS,S/AS01 response was assessed. About 50μl of peripheral blood was collected from participants in Begoro and Cape Coast in 2014, 2015, and 2016, dried blood spot (DBS) prepared and DNA was extracted. The C-terminal of Plasmodium falciparum CSP and the human leukocyte antigen (HLA) class II gene in humans were deep sequenced. The translated amino acid haplotypes of the CSP were aligned and compared to the reference 3D7 vaccine strain. The HLA class II haplotypes were grouped into superfamily and their association with the CSP variants was ascertained. The CSP haplotypes are evenly distributed between Begoro and Cape Coast. There were 31 Th2R haplotypes in Begoro and 30 Th2R haplotypes in Cape Coast; 15 Th3R haplotypes in Begoro and 13 in Cape Coast. About 83.9% of Th2R and 96.5% of Th3R haplotypes in Begoro are shared with Cape Coast. The amino acid changes with reference to the 3D7 vaccine strain at the Th2R epitope range from 1 to 6 and 1 to 4 at theTh3R epitope. There is a 53% and 60% reduction in the 3D7 Th2R and Th3R haplotypes, respectively, from 2014 to 2016, but 3D7 is still common in Ghana, Kenya, and Malawi. The 3D7 haplotype does not correlate with HLA-DRB1, but there is with HLA-DQA1 and HLA-DPB1. Begoro and Cape Coast are two different ecological zones in Ghana but the parasite population is homogenous. The Th2R epitope of CSP is polymorphic than the Th3R epitope and this higher polymorphism is driving a higher non-synonymous amino acid substitution at the Th2R epitope than the Th3R epitope which may have vaccine implication. A decline in frequency of 3D7 parasite population may also affect the performance in the vaccine in Begoro and Cape Coast. Initial correlations indicate that HLA-DPB1 (01:01/17:01) correlates with the 3D7 vaccine strain, but HLA-DPB1 (01:01/17:01) and other variants of HLA-DQA1 also correlates with other Th2R haplotypes and may compete with the vaccine haplotype for antigen presentation to CD+4 T cells. This may have implications for the efficacy of the RTS,S/AS01 vaccine in Ghana.Item Immunological Characterization Of A Novel Merozoite Surface Protein (PFMSP11)(University Of Ghana, 2020-12) Asiedu, K.Background: Plasmodium falciparum merozoite surface protein 11 (PfMSP11) is a novel surface protein that plays a critical role during the parasite’s blood stage development. This project aimed at immunologically characterizing the Plasmodium falciparum Merozoite Surface protein (PfMSP11). Method: Antibody (IgG) responses from 283 child clinical malaria samples Accra, Kintampo and Navrongo which represents different transmission intensities, were measured against the 20 synthetic peptides of PfMSP1 using ELISA. Overall 27 synthetic peptides were tested against PBMCs from 10 apparently healthy subjects to measure IFN-γ responses using ELISpot. Results: Peptides at the C-terminus of PfMSP11 gave higher antibody responses than peptides at the N-terminus. Peptide 2 to Peptide 12, and Peptide 17 recorded low antibody responses across all sites. Peptide 16 recorded the highest antibody responses across all sites. One subject (M14) showed a highest response across all pools in measuring IFN-γ responses . Subject M9 tested positive for pools 1 and 3 while subject M10 and M12 tested positive for pool 2Peptide 7 in pool 2 against subject M10 was the only peptide that tested positive. In conclusion, this study has shown that there are more antibody epitopes at the C-terminus than the N-terminus and more immunogenic T cell epitopes at the N-terminus.Item Emergence Of HIV-1 Drug Resistance Mutations in Patients After 12 Months of Antiretroviral Therapy(University Of Ghana, 2021-07) Asamoah, B.The United Nations Program on HIV and AIDS (UNAIDS) in 2013 set an ambitious target of 90-90-90, thus 90% of all people living with HIV must have been diagnosed, 90% of those diagnosed put on treatment and 90% of the patients on treatment must achieve viral suppression. The commonest cause of therapeutic failure in HIV patients is the presence of drug resistance mutations. This leads to viral rebound, reduction in CD4 count, and predisposes the patients to opportunistic infections. In addition, chances of transmission increase as a result of increased viral load. Occasionally, viruses carrying drug resistant mutations are transmitted from one person to the other. This study sought to investigate the emergence of HIV drug resistance mutations in protease and reverse transcriptase genes, twelve months after starting antiretroviral treatment.Item Membrane Vesicles Of Mycobacterium Ulcerans And Their Role In Buruli Ulcer Pathogenesis(University Of Ghana, 2021-01) Osei, E.N.The release of bacterial extracellular membrane vesicles (EMVs) is essential for pathogen’s adaptation and virulence. Mycobacterium ulcerans, the causative agent of Buruli ulcer, remains with queries in its pathogenic mechanism. The current study interrogated biological functions and protein content of EMVs from viable M. ulcerans’ cells as potential medium of virulence in BU pathogenesis. Here, we demonstrate release of intact EMVs from the thick cell wall of log-phase M. ulcerans (Nm 209) as well as M. marinum (Sa 200695) in respective liquid cultures. Size distributions of isolated EMVs were similar between the two strains and did not differ from EMVs released by M. smegmatis used as a positive controlled strain. Mycolactone could not be detected in isolated EMVs from M. ulcerans (Nm 209). However, presence of M. ulcerans EMVs was associated with higher total intracellular reactive oxygen species which eventually compromised viability of RAW264.7 cells through oxidative stress. After 48 hours of co-incubation, native and UV-A irradiated EMVs induced 45% and 40% loss in viability of RAW264.7 cells, respectively. Moribund phagocytes exhibited apoptotic changes. Proteomic analysis on the isolated M.ulcerans EMVs revealed an enrichment of 32 unique proteins mostly localized in the pathogen’s cell wall/membrane. A conserved hypothetical protein (MUL_2313), had the highest log2 fold change (11.92) followed by Amidase amiC, a cell-wall remodeling hydrolase (4.19). Others included integral membrane indolylacetylinositol arabinosyltransferase EmbA/B, and many conserved hypothetical proteins. Direct contributions of these proteins to EMVs cytotoxicity could not be established. Yet, protein moon-lighting or possible cross-linking could have potentially contributed to EMV-associated toxicity on RAW264.7 cells. Our results suggest that M. ulcerans EMVs can elicit toxic response from host’s macrophage cells through yet to be established mediators. This potentially reveals new dimension on macrophage-M. ulcerans interactions with possible contribution to local immunosuppression in BU and paradoxical reactions observed in its treatment.Item Investigation Of The Electrochemical Properties of Hydroxyapatite Immobilization Material for Potential Cytosensor Fabrication(University of Ghana, 2020-10) Adusei, D.Biomedical diagnostics is moving towards an effective and rapid diagnosis of which biosensor fabrication is the paradigm shift. Despite the successes obtained using sensing platforms, detachment of biorecognition elements from the transducing surface remains a hurdle to overcome. Good attachment of biorecognition elements to the transducing surface determines the sensitivity and specificity of the biosensor. In the area of cancer biosensing, gold, graphene, chitosan, and conducting polymers are among the few materials that have been exploited for effective immobilization, but they faced detachment problems. To curb these detachment problems, blends of cancer immobilizing materials and other molecules have been proposed but fabrication methods make the immobilizing material expensive. Thus, this thesis aimed at investigating the use of cost-effective hydroxyapatite (HAp) material synthesized from Achatina achatina snail shells (SHAp) for the direct immobilization of cells. SHAp was mixed with 3,4-ethylene dioxythiophene: poly 4- styrene sulfonate (PEDOT: PSS), a conductive polymer to increase the electrochemical responses of the SHAp forming a SHAp/PEDOT: PSS blend. The SHAp/PEDOT: PSS blend was used to modify a screen-printed electrode (SPCE) by a dropped coating approach after which cell-lines including pheochromocytoma (PC-12), human embryonic and kidney cells (HEK-293T) immobilized on the modified SPCE. Red blood cells (RBC) were used as a control. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed to record the cell proliferation signals. The CV results showed low peak currents for cell-lines (50 µA for HEK-293T and 120 µA for PC-12) and high peak current for the control RBC (230 µA). The EIS showed impedance values of 0.70 and 0.62 mΏ for HEK-293T and PC-12, respectively, and 0.52 mΏ for RBC. The findings demonstrate that SHAp is able to differentiate the proliferation signals of cells through potentiometric and impedimetric measurements. The unique current difference among these cells could be used as potential markers for the rapid detection of cancer cells at a low cost in future studies.Item Anti-Diabetic And Probiotic Effect Of Kombucha On Alloxan-Induced Diabetic Rats(2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. Kombucha was found to perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Development and Quality Evaluation of Pre-Gelatinized Instant Weaning Foods Based on Cereals and Legumes(University of Ghana, 1991-07) Abotsi, S.K.Item Development of Precooked Foods Process and Product Evaluation(University of Ghana, 1983-06) Masopeh, E.A pre-cooked food has developed from cornmeal and cowpead flour . The cowpea seeds were germinated and also dehulled. Control seeds of these treatments were made. Method used in the preparation of the product was steaming and traditional roasting in the earthen- ware mashing boul. Chemical analysis on the products Showed that the product developed had a higher protein content than cornmeal . Evaluation of the functional characteristics revealed that the water absorbed by the products increased with an increase in the number of germination days . The undehulled products also absorbed water and swelled more than the dehulled products. This ws attributed to the presence of the seed coat in the undehulled sead products. There was not much difference in the water absorption and swelling properties with respect to the levels of corn and cowpea. Viscoamylograph runs showed no change in the gelatinisation of the germinated seed products , however there was a little increase in the viscosity of the ungerminated seed product during the holding and cooling sections. Sensory evaluation of the corn-cowpea product showed that the product was acceptable. Anaalysis of variance calculations showed a significant (p~ 0.01) effect of dehulling and cersination on the colour, odor, flavor and tho beneral acceptability of the nroducts The panelists could detect differences in the different products subjected to the different treatments. A second product similar to the corn-cowpea product was developed from corndough. Fermented and unfermented corndough were used. Functional characteristics analysis showed a high water absorption and swelling of unfermented corndough products. Analysis on the product Showed no significant difference in the colour, odor etc . of the fermented and unfermented corndough products . However, the unfermented corndough product was more acceptable.Item Transmitted Drug Resistance and Co-Receptor Tropism of Circulating HIV-1 Subtypes in Art-Naïve HIV Patients in Ghana(University of Ghana, 2021-06) Quansah, D.N.K.Combination Anti-retroviral therapy (ART) has significantly reduced the burden of human immunodeficiency virus (HIV-1) infection, transforming it from a fatal disease into a manageable chronic one. The clinical benefits of ART are sustained viral suppression increase in CD4 T lymphocyte counts and improved general well-being. However, the emergence of drug resistance poses a strong challenge to the success of ART in managing HIV infection. Drug resistance (DR) to anti-retrovirals (ARVs) is caused by the high replication capacity of HIV, its error-prone reverse transcriptase and poor compliance by HIV-infected people on ART. In Ghana, reverse transcriptase (RT) and protease inhibitors (PIs) are the main drug combinations. While the transmission of multi-drug resistant variants, determined by genotypic tests, among other HIV-endemic populations informs the choice of drugs in their regimen, clinical management of HIV-1 in Ghana does not include genotypic tests. In this research, we hypothesized that transmitted drug resistant mutants present in treatment-naïve individuals are related to the co-receptors used for cell entry and may hasten ART failure. Sixty-nine treatment-naive HIV-1 infected persons were enrolled from three hospitals in Accra and their clinical histories were obtained. CD4 lymphocytes and HIV-1 viral load were determined for all samples. HIV-1 RNA was extracted from patients’ plasma and HIV Pol region was amplified by conventional PCR and sequenced. The assembled, edited and aligned contigs were used to phylogenetically characterise circulating HIV-1 subtypes. The sequences were submitted to the Stanford University HIV-DR database for analysis of drug resistance mutants. HIV co-receptor preference was measured using TZM-bl indicator cells. The dominant HIV-1 subtype was CRF02_AG (64%), followed by subtype B (26%,) and others, CRF06_cpx, A, C and G, in smaller proportions. This confirms previous reports of CRF)2_AG as predominant subtype and indicates a growing population of the subtype B, which drives the HIV epidemic in Europe and America, in Ghana. Seven mutations on the WHO drug surveillance list were present in nine out of sixty-nine participants enrolled in this study. All seven of them were either non-nucleoside reverse transcriptase (NNRTI) or PI mutations. Quite surprisingly, no nucleoside reverse transcriptase (NRTI) mutations were found. The K103N, which strongly reduces susceptibility to nevirapine and efavirenz was detected in one individual. All PI mutations that were detected were accessory mutations. This was expected since protease inhibitors are not used on a large scale in Ghana. No one individual carried more than one mutation; indicating low levels of transmitted DR among the study population The study participants had mean viral load and CD4 counts of 137,694copies/ml and 409cells/μl respectively and presented no symptoms of disease. Such attributes are indicative of rapid viral replication which is a major driver of drug resistance. Preliminary analysis of the results from the phenotypic assays showed no association between co-receptor phenotypes and identified drug resistance mutations in individuals who carried these mutations. More experiments are needed to confirm these preliminary results. This study has confirmed the predominance of HIV-1 subtype CRF02_AG as the predominant subtype in Ghana while documenting an increase in the circulation of Subtype B. Transmitted HIV drug resistance is still low in Ghana, and this is good for the country’s HIV control programmes. However, continuous surveillance for drug resistance strains among treatment-naïve HIV persons is highly recommended to inform treatment regimens going forward.Item Correlates of Virus Release in HIV-1(University of Ghana, 2019-03) Kissi-Twum, A.A.Integration of HIV-1 within the host genome may result in latent infection, where a replication competent provirus remains in a state of transcriptional silence. The latent reservoir remains the major barrier to cure efforts as to achieve cure, the reservoir should be depleted of all replication competent proviruses. Various assays to measure the latent reservoir rely on virus release and vary widely in their estimations. Quantitative viral outgrowth assay (QVOA) which is the gold standard relies on reactivation of latent proviruses to release virions and thus falls short when proviruses are non-inducible. The correlates of virus release per cell were examined in this study. The TZ-5 zipcoded library which is a Jurkat cell line with one uniquely tagged HIV-1 integrant per cell was used in this study. The library consisted of infected cells expressing HIV-1 gag and pol with an inactivated env gene and vpr-. GFP is expressed as a Nef spliced product to indicate HIV-1 expression. Intracellular Gag staining coupled with the relative amounts of p24 detected in culture supernatant showed GFP expression is an accurate proxy for HIV-1 expression. High-throughput sequencing was carried out on PCR amplified zipcodes. The topmost 74 clones which represents ≥ 97% of the raw sequence reads was analysed in this study. Although cell proliferation is thought to drive HIV-1 persistence, clonal abundance was not found to correlate with high virus release. Rather a higher GFP+ expression as determined by the green proportion correlates with high virus release per cell. Spliced and unspliced mRNA products are needed to productively assemble virions in HIV-1 although Gag which is an unspliced product is the driving force. Clones with higher amounts of unspliced product to spliced had higher green proportions and subsequently overlapped with higher amounts of virus released. Taken together, higher green proportions and unspliced RNA may predict a higher amount of virus released per cell. As green proportion is a measure of overall HIV-1 expression by a clone, incorporating the extent of HIV-1 expression in QVOA measurements will calibrate HIV-1 expression among inducible and noninducible cells in the latent pool and ultimately the decay rate of such clones. The frequency of formation of host-virus read-through and read-in chimeras and packaging into virions was examined as these unusual species can be helpful in integration site determination and ultimately determine genetic correlates of chimeric RNA generation. RNase protection assays showed the presence of chimeric read-through and read-in RNAs both in cells and virus. The genomic context of integration may contribute to our observed findings as more read-in RNA was observed to be produced and packaged into virions than read-through RNA.Item Sensitivity of Asexual Blood Stages Of . Plasmodium Berghet (Nk 65) to Chloroquine(University of Ghana., 1994-03) Aryee, N.A.The erythrocytic developmental cycle of Plasmodium berghei can be conveniently divided into the ring, tropho-zoite and schizont stages based on morphology. The sensitivity and effect of chloroquine on density-gradient isolated fractions of each of these stages was investigated using the plasmodial strain P.berghei (NK65), a rodent model as an experimental tool. This plasmodial strain was found to be routinely lethal in infected mice in the absence of administered therapeutic levels of chloroquine. P.berghei infected blood was separated into the various developmental stages using discontinuous Percoll gradient centrifugation. The various stage-isolated fractions were found to be infective and sensitive to these same levels of chloroquine. However chloroquine was found to show insignificant differential sensitivity with regards to the developmental stage of berghei (NK 65) strain.Item Molecular Subtyping and Gene Expression Analysis of Drug Resistant Invasive Non-Typhoidal Salmonella(University of Ghana, 2019-07) Adjei, R.O.Invasive Non-Typhoidal Salmonella infections have become widespread in sub-Saharan Africa. Predominant hyper-virulent clone S. Typhimurium ST313 is found to be associated with bloodstream invasion and multidrug resistance. Despite its importance, limited information is available on the circulating genotypes causing invasive non-typhoidal salmonellosis in Ghana. A total of fifty-one NTS isolates from pediatric blood samples was used for this study. The resistance profiles of isolates were assessed against 8 classes of antibiotics by disc diffusion method followed by PCR detection of resistance markers. Molecular detection of the presence of a virulence associated gene, st313-td, was used as plausive test to select isolates for Multi-Locus Sequence Typing (MLST) analysis. Additionally, the mechanism of ciprofloxacin resistance was explored by monitoring changes in expression levels of important targeted genes including gyrA, acrA, ydhJ, emrD, and soxR under drug pressure using reverse transcription quantitative PCR. Antibiotic susceptibility testing results showed all isolates were completely susceptible to imipenem, ceftriaxone and ceftazidime. In most cases, multidrug resistance (including reduced susceptibility to fluoroquinolones) were frequently recorded among S. Typhimurium isolates (63%, n=32/51). On the contrary, nine out of ten S. Dublin and the five unknown serovars were susceptible to all antibiotics used for screening. The virulence gene st313-td was detected in only 27% (n=14/51) isolates. Eight (8) other isolates showing varying resistance phenotypes were selected in addition to the 14 positive isolates for typing and analyses by MLST. Out of twenty-two (22) selected isolates, 82% (n=18), 4% (n=1) and 14% (n=3) were S. Typhimurium ST313, S. Typhimurium ST19, and S. Dublin ST 10 respectively. Most importantly, it was observed that about 83% (n=15/18) of S. Typhimurium ST313 clones were multidrug resistant (MDR). Likewise, ten selected isolates for PCR analyses tested positive for the resistance markers; CatA1 (30%), StrA (90%), BlaTem (50%), TetA (30%) & TetB (10%), Sul1 (80%) & Sul2 (100%) and aadA (70%). The normalized transformed mRNA transcript levels showed significant variations in gene expression of gyrA, acrA, ydhJ, emrD and soxR among susceptible, intermediate and laboratory induced resistant isolates. Further, sequence data analysis detected a single nucleotide polymorphism at position 324 resulting in a mutation (Phe → Asp) in the gyr A of clinical intermediate isolate, although there were no significant mutations in acrA, ydhJ, emrD and soxR genes among the three test isolates. From the results, high frequencies of multidrug resistant invasive NTS pathogens carrying plasmid-borne resistance gene markers were found. Also, reduced susceptibility to fluoroquinolones and cephalosporin were observed which calls for increased surveillance and management of antimicrobial resistance to guide treatment. Detection of a SNP in gyrA gene and variations in mRNA levels suggest possible genetic and transcriptional regulation mechanisms employed by Salmonella pathogen to withstand drug stress. Therefore, further investigations are required to understand these mechanisms which will assist in developing alternative therapeutic interventions.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 Characterizing Pathogenic Risk Factors Associated with Breast Cancer in Ghanaian Women(University of Ghana, 2019-07) Buckman, M.A.Globally, the second leading cause of cancer death in women is breast cancer. It is responsible for 16% of all cancer cases and the most common cancer in Ghana. Breast cancer pathogenesis has not been fully elucidated but is however known to have a complex aetiology involving both genetic and environmental factors. Pathogens are one of the environmental factors that have been linked with breast cancer development. Epstein-Barr Virus (EBV), Human Papilloma Virus (HPV) and Mouse Mammary Tumour virus (MMTV) have been implicated in breast cancer. Research has showed that the local microbiome of the host could modulate breast cancer risk, yet it is unknown what microbes (pathogenic or probiotic) inhabit breast tumour tissues in Ghana. The objectives of this study were to detect the presence of HPV, EBV, MMTV and bacteria in breast tumour tissues from Ghanaian women and determine the effect of bacteria on DNA in HeLa and MCF-7 cells. Formalin-Fixed Paraffin-Embedded (FFPE) tissues and fresh breast cancer tissues were obtained from 204 breast cancer patients at the Department of Surgery, Korle-bu Teaching Hospital. Nucleic acid was extracted from the samples and amplified using standard Polymerase Chain Reaction (PCR) to detect the viruses. HPV-positive tumours were sequenced using the Sanger sequencing method. EBV typing of EBV positive samples was done by a nested PCR reaction using EBV-1 and EBV-2 specific primers. Bacteria from fresh breast cancer tissues were obtained and identified using basic biochemical tests and Sanger sequencing. The DNA damage potential of the isolates was tested on HeLa and MCF-7 cells using the histone-2AX phosphorylation assay. HPV was detected in 27 (13.2%) of the 204 cases analyzed. EBV was identified in 66 (32.4%) of the samples. Out of the EBV positive cases, 2 (1%) were positive for EBV-1, 30 (14.7%) were positive for EBV-2, 26 (12.7%) were positive for both EBV-1 and EBV-2 and 8 (3.9%) could not be genotyped using available methods. Co-infection of both HPV and EBV was detected in 11 (5.4%) of the cases. MMTV was not detected in any of the samples analyzed. Microbiome analysis showed relatively high evidence of bacteria belonging to the Staphylococcus and Bacillus species. Staphylococcus sciuri, Staphylococcus epidermidis, Staphylococcus lugdunensis obtained from breast tumours induced DNA double-strand breaks in MCF-7 cells. The diversity and the probable role of the microbiome in breast cancer were also identified. Therefore, the presence of these pathogens showed probable involvement in breast cancer carcinogenesis.Item Hepatitis Delta Virus (HDV) in HIV/HBV Co-Infected Patients in Ghana(University Of Ghana, 2019-07) Attiku, K.O.Within persons infected with HIV worldwide, almost 10% have chronic HBV infection, and about 6% of all HBV infected patients are co-/superinfected with HDV, resulting in accelerated progression of hepatitis towards end stage liver disease. Even though within HIV/HBV infected patients, an increase in HDV infection has been observed, there is inadequate information on HDV prevalence as well as virologic profile in Ghana. This study sought to determine the presence of HDV in HIV/HBV co-infected patients in Ghana. This was a longitudinal purposive study which enrolled 113 patients, who have been co-infected with HIV and HBV, attending clinic at Korle-Bu Teaching Hospital (KBTH) in Ghana. Patient demographic information was obtained using a questionnaire, and 5 mL whole blood collected at two-time points (baseline and 4-6 months afterwards). The sera obtained were tested to confirm the presence of HIV, HBV antibodies/antigens and HBV DNA. Antibodies and viral RNA were also determined for HDV with respective serological and molecular assays. Amplified HBV DNA and HDV RNA were sequenced with the Sanger method and sequence reads analyzed using Sequencher software version 2.3. Phylogenetic analysis was carried out with references from the GenBank to establish the genotypes using the MEGA X software. Out of 113 samples tested, 63 (55.7%) were females and 50 (44.25%) were males. Patients enrolled in this study were within the age range of 24 to 73 years, with a median age of 45 years. A total of 100 (88.5%) samples had detectable HBV surface antigen (HBsAg), and 32/113 had detectable HBV DNA. Nucleotide sequences were obtained for 15 samples, and phylogenetic analysis showed the circulating strains to be HBV genotype E. Of 13 samples that were HBsAg unreactive, 4 (30.8%) had detectable HBV DNA and suggest the presence of an occult infection. HDV was detected in four samples and giving a prevalence of 3.54% in the HIV/HBV cohort used for the study. Two of the HDV positive samples sequenced were HDV genotype 1. In summary, the data suggest the presence and circulation of HDV and occult HBV infection in HIV patients co-infected with HBV in Ghana. The occurrence of occult HBV infection and HDV in the study population is supported by other research studies, and makes it imperative to look out for the presence of HDV and occult HBV in patients co-infected with HIV/HBV.Item Anti-Diabetic and Probiotic Effect of Kombucha on Alloxan-Induced Diabetic Rats(University of Ghana, 2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Anti-Diabetic and Probiotic Effect of Kombucha on Alloxan-Induced Diabetic(University of Ghana, 2019-07) Adade, E.E.Diabetes mellitus, is a metabolic disorder caused by the inability of the beta pancreatic cells to adequately produce insulin or due to insulin resistance of cells. As a result of the increasingly high incidence of diabetes globally, the World Health Organization (WHO) has set timelines and guidelines for the reduction of the risk of mortalities and morbidities associated with non-communicable diseases including diabetes, by the year 2030. However, this agenda is hinged on the availability of affordable, safe and effective alternatives for the management and treatment of these diseases. Hence, there is a need to explore other alternatives to the conventional oral anti-hyperglycemic agents driven by factors such as patient’s preference, demand among others. Kombucha is tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). Consumers of Kombucha have reported several anecdotal evidences of its medicinal potential. This study seeks to investigate its anti-diabetic and probiotic effect on alloxan-induced diabetic rats. It was hypothesized that Kombucha, being a complex matrix of microorganisms and nutraceuticals, would play an essential role in the management of diabetes. Molecular characterization of the microbiome of Kombucha using shotgun metagenomics (Oxford Nanopore MINION sequencing technology) showed Brettanomyces bruxellensis CBS 2499 as the most abundant species within the microbial community accounting for about 51 % of all reads. Brettanomyces anomalus, Komagataeibacter xylinus NBRC 15237, Bacillus nealsonii AAU1, Zygosaccharomyces bailii CLIB 213, Acetobacter, Gluconobacter and over 300 other genera and species of microorganisms including archaea and viruses were also detected using a combination of REFSEQ and One Codex data bases (OXCDB). In-vivo experiment was used to evaluate the anti-diabetic property, safety and gut microbiome changes of Kombucha. Kombucha was found to perform better than the conventional antidiabetic drugs, metformin and glibenclamide in lowering the fasting blood glucose (FBG) of the diabetic rats. Daily administration of 25 mg/kg and 100 mg/kg of freeze-dried Kombucha tea demonstrated a 5 fold reduction in FBG (p<0.05) and 40% and 50% respective increases in body weight of the alloxan-induced diabetic rats compared to the diabetic control (DC). Histological analysis, shows Kombucha enhances pancreas regeneration and hence the concomitant increase in insulin secretion as demonstrated in the study. Serum lipid profiling showed 100mg/kg Kombucha treatment increases the levels of total cholesterol (16%), high density lipoproteins (HDL) (13%) and low-density lipoproteins (LDL) (10%) but conversely reduces triglyceride level (17%) compared to the DC (p>0.05). Further analyses demonstrated that Kombucha decreases the relative organ (liver and kidney) to body weight ratio in treated animals. In addition, Kombucha was able to reduce significantly the elevated levels of liver enzymes such as Alkaline phosphatase (ALP), Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) as well as renal toxicity indices, creatinine and urea in treated animals. Histology of the kidney and liver also showed that Kombucha has no adverse effect on the morphology and cellular integrity of these organs suggesting its hepatoprotective and renal protective potentials. Urinanalysis also showed reduction of glucose in urine for the 100 mg/kg Kombucha-treated animals. Additionally, Kombucha protects the gut microbiome, most significantly by enhancing the Lactobaccillaceae family of bacteria within the gut and reduces the possibilities of colonization of the gut by other opportunistic bacterial species. The study demonstrated that Kombucha is enriched with diverse microbial population with probiotic value and daily intake of Kombucha may be potentially helpful in the management of diabetes, protection against renal and liver toxicity and offer gut microbiome protection.Item Identification of Natural Product-Derived Compounds that Inhibit Hiv-1 Entry into Target Cells(University of Ghana, 2019-07) Ugwu, N.P.HIV-1 infection is mediated by the interaction between the host cellular CD4 receptor and co-receptors with HIV Envelope protein (Env). Inhibition of HIV entry into host cell presents a biological target for therapeutic intervention. Broadly neutralizing antibody (bNabs) are potent in neutralizing a broad range of HIV strains by binding to the Env and preventing entry into the host cell. VRC01 is a CD4 binding-site class bNabs that binds to the conserved CD4 binding region of Env. However, using antibodies as therapeutic agents pose challenges of low availability and high cost of production. Natural products that can mimic VRC01 bNabs by interacting with the conserved CD4-binding regions may serve as new generation of HIV-1 entry inhibitors, which are broadly reactive and potently neutralizing. This study aimed to identify natural products that mimic VRC01 broadly neutralizing antibody, by interacting with the CD4-bs of HIV-1 gp120 and inhibit viral entry into a target cell. A library of purchasable compounds derived from natural products was virtually screened against the HIV-1 gp120 of clade A/E recombinant 93TH057 (PDB: 3ngb) using AutoDock Vina, and LIGPLOT was used to elucidate the interactions between the compounds and the HIV-1 gp120 complexes. The compounds with the lowest binding energies were selected and further screened against gp120 of a reference HIV strain (HXB2). Pharmacological profiling of the compounds was undertaken using SwissADME. Ten selected compounds were purchased and used for cell-based antiviral infectivity inhibition assay using HXB2-env pseudotype virus. Three compounds, NP-005114, NP-008297 and NP-007382 inhibited entry of HIV HXB2 pseudotype virus into target cells, while the rest of the compounds showed no inhibitory activity against HIV entry into target cells. NP-005114, extracted from the seed of T. chebula, had IC50 of 10μg/ml. Protein-ligand interaction showed that NP-005114 had inter-molecular hydrogen bond interactions with eight and eleven amino acid residues on the CD4- binding site of recombinant clades A/E and B HIV -1 gp120, respectively. NP-005114 had intermolecular hydrogen and hydrophobic interactions with Asp457, a critical amino acid residue in the Phe43 cavity of the HIV gp120 for recombinant clades A/E and B, respectively. Derived from the leaf of Ginkgo biloba, NP-008297 formed ten and four hydrogen bond interactions with amino acid residues of the CD4-binding site on HIV-1 gp120 of recombinant clades A/E and B, respectively. NP-008297 inhibited viral entry at IC50 of 15.5μg/ml. NP-007382 was derived from bacteria and interacted with five amino acid residues in the CD4-binding site of HIV-1 gp120 of recombinant clades A/E and B, via intermolecular hydrogen bond. The 50% inhibitory concentration of NP-007382 against viral entry into the target cell was 13.1μg/ml. These compounds inhibited the entry of HIV clade B pseudotype (HXB2) with an IC50 which was comparable to that of VRC01 (0.1-50 μg/ml) against HIV clade B pseudotypes and primary isolate. The interaction of the compounds with critical residues of the CD4-binding site of more than one clade of HIV-gp120 demonstrates the possibility of broad entry inhibition of other HIV clades. This is the first report of the characterization of NP-008297, NP-00738, and NP-005114 as HIV-1 entry inhibitors.