Department of Biochemistry, Cell and Molecular Biology

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    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.
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    In Vitro Studies Of The Effect Of Anopheles Gambiae Midgut Bacteria On The Development Of Plasmodium Falciparum
    (University Of Ghana, 2021-12) Ametsi, Williams Godwin
    During 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.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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.