Department of Biochemistry, Cell and Molecular Biology
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Item Utilizing Yeast as a Model Organism for Drug Discovery against Eukaryotic Infections(University Of Ghana, 2017-07) Abban, R.L.Eukaryotic pathogens are responsible for several serious human diseases, however, most drugs used as treatment regimens are ineffective while others still in use are toxic. This study seeks to identify bioactive molecules from wood decay fungal (WDF) extracts with activity against eukaryotic pathogens (especially Candida albicans and Plasmodium falciparum). The Saccharomyces cerevisiae model organism for eukaryotic cell biology studies is extensively used to screen bioactive molecules because it allows for rapid. Twelve WDF cultures were extracted with ethyl acetate, and the extracts were screened against C. albicans and S. cerevisiae by the disc diffusion method. Phenotypic array assays were also conducted on selected wood decay fungal extracts. The 3 most active extracts were then fractionated via bioactivity guided fractionation using different chromatographic techniques (solvent partition chromatography, size-exclusion chromatography and preparative TLC). The antifungal and antiplasmodial activities of the fractions were confirmed using MTT assay and SYBR Green 1 growth inhibition assays respectively. The phenotypic array assays performed on the selected wood decay fungal extracts showed different responses to C. albicans and S. cerevisiae, and this led to the selection of top the 3 most-effective extracts as the best candidate for natural product isolation. After an extensive bioactivity-guided fractionation, 49 out of a total of 125 2D Preparative TLC fractions showed antifungal activities in the MTT assay while 47 of the 55 second dimensional preparative TLC fractions showed antiplasmodial activities in the SYBR Green 1 growth inhibition assay. These findings define wood decay fungi as a viable source of antifungal and antiplasmodial compounds, which can be pre-selected using S. cerevisiae over hemolysis problems posed by the total fungal extracts. It will take larger volumes of fungal cultures to finally isolate the many active compounds for structural development and possible clinical development.Item Discovery of Buruli Ulcer Infection Associated Metabolic Markers(University of Ghana, 2017-07) Laryea-Akrong, E.K.Buruli ulcer (BU), is a severe slow progressing necrotizing skin disease, caused by the environmental pathogen Mycobacterium ulcerans (MU). The diagnosis of the disease can be carried out in four main ways. These include microscopy which is preceded by Ziehl-Neelsen stain, PCR amplification of the IS2404 insertion sequence, histopathology and culture. The limitations of BU diagnosis include microscopy which is not sensitive and specific, culture which takes 8-12 weeks for a growth to be observed and PCR which requires skilled labour and is very expensive. Early diagnostic techniques are urgently needed for this disease at areas and communities where they are most endemic since the disease can be treated if it is diagnosed early. The aim of this study was to discover Buruli ulcer infection associated metabolic markers using a metabolomics approach. Wound swabs from 47 participants were taken and BU cases were confirmed with microscopy using Ziel-Neelson staining and PCR. Metabolites were extracted from the wound swabs with chloroform, methanol and water and derivatized for Gas Chromatography-Mass Spectrometry to determine the different metabolites of patients with or without the infection. After DNA extraction and PCR 65% of the cases were found to be positive for mycobacterial DNA, 34% of the cases were also found to be positive for M. ulcerans and 8.5% were found to be positive for H. ducreyi. Seventeen metabolites were identified, and palmitate was found to be significantly higher when the cases were compared to the controls. Palmitate and stearate were found to show significance between the cases that were on treatment and those that were not on treatment. For all cases that were positive for mycobacteria they had significantly higher levels of pentadecanoate and heptadecanoate. Finally, cases positive for H. ducreyi and had higher levels of cervonate (docosahexaenoic acid) and palmitate. Unique metabolites specific to the BU cases were found and these can be confirmed as biomarkers and utilized in the development of a possible diagnostic for this Neglected Tropical Disease.Item Characterization of Liver Function, Immune Response And Hepatitis B Virus (Hbv) Genotypes in Pregnant Women With Malaria-Hbv Co-Infection in Northern Ghana(University of Ghana, 2017-07) Anabire, N.G.Background: The overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, the impact of this co-infection on liver function and immune responses is unknown. This study sought to investigate these interactions in pregnant women reporting to antenatal clinic in the Northern Region of Ghana. Methodology: Levels of malaria parasitemia, hepatitis B viremia, liver biochemical parameters and inflammatory cytokines were assayed and compared across four categories of pregnant women: uninfected, infected with Plasmodium falciparum alone (Malaria group), infected with hepatitis B virus (HBV) alone (CHB group) and co-infected with P. falciparum and hepatitis B virus (Malaria+CHB group). Circulating HBV genotypes were determined by nested PCR. Results: the prevalence of HBV genotypes were: 91.6% genotype E, 5.2% mixed genotypes E/A, and 3.2% mixed genotypes E/D. Relative to the CHB group, the Malaria+CHB group had lower viremia levels (P=0.0157). However, levels of malaria parasitemia were similar in women in the Malaria and Malaria+CHB groups (P=0.3038). Furthermore, levels of markers for liver injury/damage, including alanine aminotransferase (P <0.0001), aspartate aminotransferase (P <0.0001) and total bilirubin (P <0.0001) were elevated in women in the Malaria+CHB group relative to those in the other groups. Similarly, proinflammatory cytokines, including tumour necrosis factor alpha (TNF-α) (P <0.0001), interleukin (IL)-1β (P =0.0270), and IL-6 (P =0.0106) were higher in women with Malaria+CHB compared to those in the other categories. For anti-inflammatory cytokines, including IL-10 (P <0.0001) and IL-4 (P =0.0007), the pattern was exactly the opposite of that for the pro-inflammatory cytokines. Conclusions: The study reports HBV genotype E to be dominant among pregnant women in Northern Ghana. Our findings showed that malaria/CHB co-infection in pregnancy appeared to exacerbate the release of biomarkers for liver damage and inflammatory mediators while reducing immune-modulatory mediators. Recommendation: Further longitudinal studies to investigatechanges in the levels of these biomarkers after the malaria infection is cleared will be important to confirm the findings of this study.Item Analysis of Histidine Rich Protein 2 And 3 Gene Deletion Polymorphisms in Northern Ghana(University of Ghana, 2017-07) Ayelazuno, F.A.The use of Plasmodium falciparum histidine rich protein 2 based (PfHRP2-based) Rapid Diagnostic Tests (RDTs) to accurately diagnose falciparum malaria from other febrile cases reporting to health facilities in Ghana plays a vital role in the control of malaria. However, false negatives due to deletion polymorphism in the pfhrp2 gene may lead to misdiagnosis, increased morbidity as a direct result of delayed treatment and ultimately high treatment cost. Therefore, determining the prevelance of parasites that carry these polymorphisms could be of relevance to National Malaria Control Programmes (NMCPs). The aim of this study was to determine the prevalence and geo-spatial distribution of P. falciparum histidine-rich protein 2 and 3 gene deletion polymorphism in the Kassena-Nankana Districts (KNDs). Patients reporting with fever or history of fever were recruited after informed consent was obtained. Thick and thin blood smears were used to assess malaria parasite species and density of parasitemia whilst filter paper dried blood spots (DBS) were made for parasite DNA extraction for detection of deletion polymorphism. DNA was extracted using Qiagen midi kit following the manufacturer’s instructions. The exon 1-2, exon 2 and the flanking genes of pfhrp2, the exon 2 of phrp3 were amplified using specific primer pairs. PCR products were resolved by 2.0 % agarose gel electrophoresis. A total of 197 samples were collected, out of which 99 were found to be positive for P. falciparum. The prevalence of parasites that were found to have deleted exon 1-2 of the pfhrp2 gene was 7.7% (7/99), whilst 14.1% (14/99) were detected to have deleted the upstream gene of pfhrp2 gene. I observed 4.0 % (4/99) of parasites that had deleted exon 2 of the pfhrp2 gene and only one parasite was found to have deleted the entire pfhrp3 gene. Geo-spatial analysis did not reveal location specific differences in prevalence of pfhrp2 deletion polymorphisms. Overall, this study shows a low prevalence pfhrp 2 and/or 3 gene delection in the KNDs of northern Ghana. Evidence that pfhrp2 based RDTsmay still be an effective tool for diagnosing malaria in this region.Item Isolation and Characterization of Haemophilus Ducreyi Strains from Children with Cutaneous Lesions in Yaws Endemic Regions, Ghana(University Of Ghana, 2017-07) Simpson, S.V.Recent discovery of cutaneous H. ducreyi has complicated the epidemiology of Yaws in endemic countries. Yaws and H. ducreyi ulcers are clinically indistinguishable from each other and some other causes of skin ulcerations. The aim of the study was to isolate and characterize H. ducreyi strains from lesions of children in yaws-endemic areas. Symptomatic patients were first screened with Dual Path Platform (DPP-RDT) Syphilis Screen & Confirm test kit (Chembio, Medford, New York) for yaws. Lesion exudates were tested by culture for H. ducreyi and real-time multiplex PCR assays were used to identify T.p subsp. pertenue DNA and H. ducreyi DNA. Azithromycin (AZT) resistance markers were screened for in T.p subsp. pertenue PCR positives. Bacterial 16S rRNA gene was amplified and sequenced to detect the presence of other pathogenic bacteria. Patient data showed 84 of 115 more males than females, mean aged 10 years. Eighty-seven percent had a clinically apparent skin lesion with few having skin conditions clinically consistent with yaws. Dual DPP-RDT positives were 64 while dual DPP-RDT negatives were 51. Of 60 bacteria culture positives obtained from symptomatic patients, 7 yielded a definitive diagnosis of H. ducreyi. Isolated cutaneous H. ducreyi strains by their appearance in colony morphology and colour differed compared to genital H. ducreyi strain, HD 35000. Out of 112 samples, 32 were H. ducreyi PCR positive, 11 were T.p subsp. pertenue PCR positive, while 1 had both pathogens. An A2058G point mutation in the 23S rRNA gene of T.p subsp. pertenue indicates resistance to AZT. A total of 69 of 112 samples with unknown aetiology could possibly be any of these bacterial species; Fusobacterium necrophorum subsp. funduliforme, Catonella morbi and Staphylococcus capitis subsp. capitis amongst others. This study suggest the need to isolate bacterial species associated with cutaneous lesions to screen for other antibiotics to be used as a combination therapy with AZT during mass drug administration (MDA) activities.Item Molecular Characterization Of Tick-Borne Parasites In Naturally Infected Cattle In Accra And Adidome, Ghana(University of Ghana, 2017-07) Adzigbe, J.Tick-borne pathogens and ticks pose significant problem to livestock production especially cattle production. This causes huge economic decline to livestock farmers in tropic and sub-tropic regions of Africa. The most economically important tick-borne pathogens to the production of bovine include Babesia, Anaplasma, Theileria and Ehrlichia. In Ghana, inadequate data exists on the species and genetic diversity of tick-borne hemoparasites. The current study was conducted to detect and characterize tick-borne pathogens and determine their genetic diversity in naturally infected cattle in two agro-ecological zones in Ghana. A total of forty cattle, twenty from each study site were sampled at four to five week interval over a duration of six months. A nested PCR amplifying the 18S rRNA gene was performed to detect the presence of tick-borne parasites. Host and environmental factors were assessed to determine whether they played a role on frequency of detection of these parasites. Species and genetic identity of the tick-borne parasites were established through sequencing of their 18S rRNA gene amplicons followed by sequence homology search at NCBI DNA data base and phylogenetic analysis of the sequenced amplicons. Theileria species and Babesia species were the two genera of tick-borne pathogens identified. T. velifera was the predominant species of tick-borne hemoparasite identified at both study locations. Also, T. mutans and B. bovis were identified at Accra and Adidome respectively. The tick-borne parasites exhibited diverged dynamics of infection throughout the period of the study. Breed of cattle significantly affected the frequency of detection of the tick-borne parasites. Environment and sex did not play a role in the frequency of detection of the parasites. There was no genetic diversity between the same species of parasites identified from the same agro-ecological zones.Item Serum Biochemical Parameters and Cytokine Profiles Associated With Animal African Trypanosomiasis in Naturally Infected Cattle in Ghana(University Of Ghana, 2016-07) Bakari, S.M.Animal African Trypanosomiasis (AAT) greatly affects livestock production in sub-Saharan Africa. In Ghana prevalence of AAT is estimated to range between 5 – 50%. Studies have reported serum biochemical aberrations and variability in cytokine profiles in animals during infection. However, information regarding the biochemical parameters and cytokine profiles associated with natural infections are limited. This study was therefore aimed at establishing the levels of serum biochemical parameters and cytokine profiles in naturally infected cattle over a period of six months in 40 individual cattle at 4 – 5 weeks intervals. Nested Internal Transcribed Spacer (ITS)-based PCR and sequencing were used to characterize trypanosome infection in cattle at two areas (Adidome and Accra) of different endemicities. Levels of serum biochemical parameters (Creatinine, Cholesterol, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Total bilirubin and Total protein) and cytokines (IL-10, IL-4, IFN-γ, TNF-α and IL-12) were measured in serum samples collected and then compared between infected cattle and uninfected controls. The predominant trypanosome species detected in Accra (non-endemic) and Adidome (endemic) were Trypanosoma theileri and Trypanosoma vivax respectively. Serum biochemical parameters were similar between infected and uninfected cattle in Accra. Infected cattle at Adidome however had significantly higher levels of ALP (infected = 613.15U/L, uninfected = 342.5U/L, p = 0.02), Creatinine (infected = 101.90 μmol/L, uninfected = 77.75 μmol/L, p = 0.01), Total Protein (infected = 77.14 μmol/L, uninfected = 71.26 μmol/L, p = 0.01) and Total Bilirubin (infected = 2.45 μmol/L, uninfected = 1.96 μmol/L, p = 0.04) and significantly lower levels of cholesterol (infected = 2.45 mmol/L, uninfected = 3.15 mmol/L, p = 0.02) at specific time points. University of Ghana http://ugspace.ug.edu.gh iv At basal levels and during infection, significantly higher pro-inflammatory/anti-inflammatory (Th1/Th2) cytokine ratios were observed in cattle at Adidome compared to Accra, indicating a shift towards Th1 immune response in Adidome. Levels of IL-10 and TNF-α were however significantly elevated in infected cattle in Accra, suggesting high anti-inflammatory cytokine response in Accra. These results generally demonstrate that cattle in the non-endemic area, which were predominantly infected with non-pathogenic trypanosome species, have different biochemical profiles and immune responses compared to cattle in the endemic area with predominantly pathogenic trypanosome species.Item Genotyping And Drug Susceptibility Testing Of Mycobacterial Isolates From A Population-Based Tuberculosis Prevalence Survey In Ghana(University of Ghana, 2016-07) Addo, S.O.Sub-Saharan Africa accounted for about 28% of the estimated 9.6 million of all notified tuberculosis (TB) cases in 2014. Due to high incidence of TB caused by Mycobacterium tuberculosis complex (MTBC) in developing countries, TB control activities have centered on MTBC to the neglect of non-tuberculous mycobacterial (NTM) infections. MTBC and NTM infections differ clinically, however, microscopy which is the mainstay of TB diagnosis in Ghana cannot distinguish between them. The inability to characterize acid fast bacilli (AFB) has led to improper treatment resulting in susceptible MTBC strains having an increased risk of developing resistance to first-line anti-TB drugs. In Ghana, very few studies have reported on the incidence and prevalence of TB due to specific MTBC species and their drug susceptibility test (DST) to first-line anti-TB drugs in Ghana. However, these studies did not have nationwide coverage. On the other hand, there is lack of national data on the species of NTM causing pulmonary infections as well as their DST patterns. This study sought to use World Health Organization (WHO) approved line probe assay (LPA) to differentiate MTBC and NTM isolates obtained from population-based TB prevalence survey in Ghana and to determine their DST patterns to isoniazid (INH) and rifampicin (RIF); and macrolides and aminoglycosides, respectively. A retrospective study was conducted whereby a total of 361 mycobacterial isolates were differentiated and their drug susceptibility patterns determined using GenoType Mycobacterium Assays: MTBC and CM/AS for differentiating MTBC and NTM as well MTBDRplus and NTM-DR for DST patterns of MTBC and NTM, respectively. All the isolates were obtained from sputum of participants aged between 15-100 years comprising 159 males and 202 females. Out of 361 isolates, 165 (45.7%) were MTBC while 196 (54.3%) were NTM. The MTBC comprised 161 (97.6 %) Mycobacterium tuberculosis and 4 (2.4%) Mycobacterium africanum. Eighteen (10.9%) and 2 (1.2%) of the MTBC isolates were INH and RIF monoresistant respectively while 11 (6.7%) were multi-drug resistant (MDR). RIF resistance was associated with D516V (46.7%), H526Y (23.1%) and S531L (15.4%) mutations in rpoB whiles INH resistance was frequently associated with S315T (82.8%) mutation in katG. Mutations in the promoter region of inhA (34.4%) also resulted in isoniazid resistance. Fourteen different NTM species were identified, majority (21.4%) being M. fortuitum. NTM-DR enabled the DST of thirty six NTM isolates belonging to the M. avium complex and M. abscessus complex. These isolates were all susceptible to macrolides (clarithromycin, azithromycin) and aminoglycosides (kanamycin, amikacin, and gentamicin). In conclusion, M. tuberculosis is still the dominant species causing TB in Ghana whiles M. fortuitum is the most frequently isolated NTM species from sputum. Also, resistance to isoniazid is high compared to rifampicin. The study showed the usefulness of the GenoType Mycobacteria assay series as appropriate tools for simple and rapid differentiation and DST of mycobacterial isolates to enhance adequate and prompt treatment of mycobacterial infections in Ghana.Item Genotypic Characterization Of HIV-1 From Patients In Two Regions Of Ghana(University of Ghana, 2016-07) Deletsu, S.D.The HIV/AIDS pandemic is still a global health issue despite the many concerted efforts put in by various stakeholders to curtail it. It has become clear however that understanding the epidemiology of the highly variant virus is key in eliminating it. It has been shown that the subtype of infecting HIV-1 plays a crucial role in disease progression among other thing. It is therefore important to continually monitor the circulating HIV-1 subtypes in the population. Equally important, is the need to monitor the occurrence of drug resistance mutations in the population. With the continuous increase in the prevalence of drug resistance HIV-1 strains, it is key to be aware of the prevalence of drug resistance in the population in order to combat it. This study aims to determine the common circulating HIV-1 subtype and to identify the drug resistance mutations present, if any, in the sampled population. Viral RNA was extracted from sixty-three (63) archival plasma samples collected from four groups of HIV-1 patients in a previous study. The HIV protease and Reverse Transcriptase genes were amplified by polymerase chain reaction and positive amplification products sequenced and analysed to determine the subtype and to identify drug resistance mutations. The protease and reverse transcriptase genes were successfully amplified from 46% and 33% of samples respectively. From these, 27.6% and 69.1% were successfully sequenced for the protease and reverse transcriptase genes respectively. All (100%) of the protease subtypes were CRF02_AG. Of the reverse transcriptase subtypes, 77% were CRF02_AG, 13% G and 10% were CRF01_AE subtype. Drug resistance mutations were identified in 37.5% of protease sequences and 53.9% of reverse transcriptase sequences. The most prevalent NRTI mutation was the M184I which translated to a high frequency of lamivudine resistant samples. About 85.7% of reverse transcriptase sequences were resistant to both NNRTIs (Nevirapine and Efavirenz) in use in Ghana. This study provides evidence that the CRF02_AG is the common circulating subtype in the sampled population and indicate the presence of high frequency of resistance to Nevirapine and Efavirenz.Item The Effects Of Cryptolepis Sanguinolenta Root Extracts On Plasmodium Falciparum Gametocyte Development(University of Ghana, 2016-04) Arku, A.T.Current efforts to eliminate malaria worldwide are soaring and one of the approaches to this is the mass screening of medicinal plants for their possible potential anti-malarial property. The current study conducted was to determine the effect of one such plant, Cryptolepis sanguinolenta on gametocyte development. In vitro growth inhibition assays on early and late gametocytes stage using ethanolic and aqueous extract of the C. sanguinolenta roots was performed. Enzyme activity assays were conducted to determine the effect of C. sanguinolenta ethanolic extract on aconitase, citrate synthase and α-ketoglutarate dehydrogenase activity in late gametocyte stages. Rhodamine 123 and bodipy-tr ceramide dye labelling of C. sanguinolenta treated gametocytes was also performed to determine the effect of C. sanguinolenta ethanolic root extract on the gametocytes mitochondrial function and the intracellular membranes integrity respectively. C. sanguinolenta inhibited gametocytes with IC50 obtained for C. sanguinolenta ethanolic and aqueous extracts on early gametocyte stages, 307 ± 47.4μg/ml and 305 ± 42.8μg/ml respectively. The IC50 obtained for C. sanguinolenta ethanolic and aqueous extracts on late gametocyte stages were 291.2 ± 24.668μg/ml and 307.3 ± 20.42μg/ml respectively. In the treated late gametocytes stage, C. sanguinolenta ethanolic extract at 90% inhibitory concentration significantly inhibited citrate synthase activity (P = 0.04) and aconitase activity (P = 0.001) but not α-ketoglutarate dehydrogenase activity (P = 0.1). Loss of uptake of Rhodamine 123 dye in the matrix of the ethanolic extract of the C. sanguinolenta treated late gametocytes stage suggested total collapse of the gametocyte’s mitochondrial membrane potential. Consequently, this led to the loss of membrane integrity of other intracellular membranes within the treated late gametocytes stage as observed from the reduced uptake of bodipy-tr ceramide dye. This study suggests that the ethanolic roots extract of the C. sanguinolenta reduced the energy production capability of late gametocyte stages, which consequently impaired their growth and therefore possible loss of their infectivity.