Academic submissions (CoVAB)

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    Genotypes of Hepatitis B Virus in Sera of patients positive for HbsAg at Jinja Regional Referral Hospital between January-April, 2019
    (Makerere University, 2022) Ssekibule, Robert
    Hepatitis B virus infection is a serious disease that manifests clinically either as an acute hepatitis or in various chronic conditions causing approximately 650,000 deaths worldwide every year. About 10% of the Uganda’s population is chronically infected and are at risk of developing severe HBV liver related diseases. The HBV exists in different genotypes and each genotype has been shown to influence its clinical out and treatment differently. There is limited data about the circulating genotypes in different regions of Uganda where HBV is highly prevalent. In this study, the circulating genotypes of HBV in patients that reported to Jinja regional referral hospital between January to April 2019 were determined. In total, 102 stored HBsAg positive serum samples obtained from patients who reported to the hospital for HBV screening and testing between January to April, 2019 were included in the study. The DNA from the serum samples was extracted and tested them with PCR using primers targeting highly conserved pre-core/core region of all HBV genotypes. Of the 102 samples, 54 were successfully amplified as HBV which were then amplified by genotype-specific primers in order to determine the genotypes in the samples. Thirty five samples were successfully amplified by the different primer sets and the HBV genotypes identified as follows: D (n=12; 37.5%); A (n=8; 25.0%); E (n=2; 5.71%); C (n=1; 2.86%); B (n=1; 2.86%) and mixed genotype infections (n=11;31.43%). Representative amplicons of the different genotypes were sequenced and phylogenetic analysis carried out. All sequences of the amplicons were in agreement with the results of genotype-specific primers with the exception of genotype C which instead clustered with genotype A. In conclusion, genotype A and D were the most dominant genotypes in patients reporting to Jinja regional referral hospital. Proper management of HBsAg positive patients is recommended since Genotype D, the most dominant genotype in the region is associated with severe clinic outcomes.
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    Evaluation of nanoparticles and phages as candidate therapeutic agents for carbapenem resistant Klebsiella pneumoniae and Escherichia coli
    (Makerere University, 2022-09) Ssekatawa, Kenneth
    Centred on the current rate of antibiotics production and approvals, it is anticipated that by 2050 up to 10 million people could die annually due to multidrug-resistant (MDR) pathogens. One of the notable examples of antibiotic resistance is the evolution of carbapenem-resistant Enterobacteriaceae. The development of carbapenem-resistance (CR) is of great concern to the healthcare systems as there is no apparent next line of antibiotics to use against carbapenemase-producing (CP) Enterobacteriaceae. Among the Enterobacteriaceae, a high prevalence of carbapenem-resistance has been reported in Escherichia coli and Klebsiella pneumoniae. Indeed, the WHO expert committee on antimicrobial resistance included carbapenem-resistant Klebsiella pneumoniae and Escherichia coli associated with the severe urinary tract, bloodstream and lower respiratory tract infections as critical priority species. The ability to cause diseases is potentiated by the presence of virulence factors. The virulence factors influence the capacity of E. coli and K. pnuemoniae to infect and colonize different body systems. The coexistence of virulence and beta-lactamase encoding genes complicates treatment outcomes. To this effect, this study investigated the carbapenem resistance profiles of pathogenic E. coli and K. pnuemoniae in Uganda. Furthermore, nanomaterials and bacteriophages as alternative approaches to treat bacterial infections were evaluated. A retrospective cross-sectional study was conducted using 421 E. coli and 227 K. pnuemoniae archived clinical isolates collected from four Uganda tertiary hospitals between January and December 2019. The isolates were subjected to antibiotics sensitivity assays to determine phenotypic resistance. Five sets of multiplex PCR were performed to detect Carbapenem-resistance genes; DEC pathotypes virulence genes; ExPEC PAI; E. coli phylogenetic groups; and K. pnuemoniae capsular pathotypes. Silver and copper oxide nanoparticles were green synthesized using plant extracts. Chitosan was isolated from the banana weevils, mushrooms and Nile perch scales by demineralization, deproteination and deacetylation. Chitosan nanoparticles were prepared using the ionic gelation method. The AgNPs, CuONPs, Chitosan, and its derivatives were characterized by UV Vis spectroscopy, XRD, FTIR, DLS, SEM, EDX and TEM. Assessment of the antimicrobial activity of AgNPs, CuONPs and chitosan was done by the agar well diffusion method. Furthermore, E. coli phages were isolated from sewage by the enrichment method. The phage host range was determined by the spotting assay. Synergism of phages and nanomaterials in the elimination of biofilms was performed on a 24 well plate. Morphological and molecular characterization of phages were conducted. E. coli and K. pnuemoniae registered statistically similar phenotypic resistance (p-value = 0.9999). Molecular screening detected carbapenem-resistance determinants in 36.6% of the MDR isolates. K. pnuemoniae isolates recorded a genotypic prevalence of 43.2%, whereas E. coli scored 33.0%. Therefore, the prevalence of the carbapenem resistance genes was significantly higher in K. pnuemoniae (p-value < 0.0001). A significant variation between phenotypic and genotypic resistance was observed; however, the presence of resistance genes highly correlated with phenotypic resistance. A high-level carbapenem-resistance carriage was observed among pathogenic E. coli and K. pnuemoniae isolates. Among the 249 pathogenic E. coli, phenotypic resistance was found in 31.7% (79) isolates. For K. pnuemoniae capsular types, 34.9% (37/106) exhibited phenotypic resistance. Accordingly, compared to E. coli pathotypes, virulence factors and carbapenem resistance co-occurrence was significantly higher in K. pnuemoniae capsular types (p-value < 0.0049). Thus, the present study reinforces that carbapenem resistance is still a significant threat in Uganda as the phenotypic and genotypic resistance observed are statistically similar to those reported by earlier studies. Two novel phages (named UGKSEcP1 and UGKSEcP2) without any undesirable genes belonging to the Myoviridae phage family, Tequatrovirus genus, exhibited lytic activity against all the detected carbapenem-resistant E. coli pathotypes and four uncharacterized carbapenem-resistant K. pnuemoniae clinical isolates with extensive clear plaques. Furthermore, chitosan isolated from locally available materials exhibited antibacterial activity at extremely high concentrations (>3000 µg/ml), whereas the biosynthesized silver and copper nanoparticles had potent antibacterial activity at low MICs. Silver nanoparticles and silver-chitosan nanohybrid significantly reduced the phage viability but did not completely inactivate. A combination of phages and nanomaterials significantly reduced the planktonic and biofilm bacterial population density than the individual components. Furthermore, we found that the cocktail of phages and nanomaterials was more effective in preventing biofilm formation than treating. As the combinatory effect of phages and the nanomaterials was significantly higher than the individual components against carbapenem-resistant E. coli planktons and biofilms, therapeutic formulations containing phages and nanoparticles should be considered as alternatives to combat antimicrobial resistance.
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    Trypanosoma Brucei Brucei : host interactions associated with biological barrier traversal
    (Makerere University, 2022) Namayanja, Monica
    Trypanosoma brucei, the causative agents of African Trypanosomiasis cross different biological barriers resulting in serious disorders. This study investigated Trypanosome parasite host interactions associated with biological barrier traversal and the mechanism used by T .b. brucei to traverse different biological barriers. A mouse model utilizing different T. b. brucei strains was employed to seek whether traversal of the blood-brain barrier (BBB) commences at the same time point for different strains of the same subspecies. Further, in vitro biological barrier models, the MDCKII and HDMEC models, together with confocal and scanning electron microscopy were used to determine the mechanism of traversal. Subsequently, a protein previously suspected to be involved in cell-cell adhesion, and a possible trypanosome ‘integrin’ was expressed both in the bacterial expression system and ectopically overexpressed in the trypanosome and later localized. Findings show that different strains of the same subspecies begin traversing the BBB at different time points and that both field isolates and laboratory strains as early as 3 days post infection, which is earlier than initially reported. Traversing of different biological barriers by T. b. brucei was found to differ, with potentials of modifying the host plasma membrane in one of the barriers as it traverses through the formation of “possible cup-like structures independent of vascular adhesion molecule-1. Both paracellular and transcellular routes of traversal are utilized though with more preference to paracellular route. The expressed protein was found to localize in the mitochondria and not on the plasma membrane, ruling out the possibility of its involvement in cell-cell adhesion or BBB traversal. Taken together, the findings of this study suggest the possible involvement of different mechanisms and different virulence factors in BBB traversal. It also for the first time demonstrates the modification of the host plasma membrane as the T. b. brucei migrates across the microvasculature. Exploring the factors behind this modification may unveil trypanosome novel molecules that can be exploited in development of new interventions against African Trypanosomiasis.
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    Detection of carbapenem resistance due to outer membrane porin alteration in non carbapenemase producing E. coli and K. pneumoniae isolates from Mulago National Referral Hospital, Uganda
    (Makerere University, 2022) Ndyagumizamu, Moses
    Antimicrobial resistance is a global public health concern which compromises the use of antibiotics in the fight against microbes. Carbapenems are the most effective antibiotics for the treatment of clinical infections caused by Enterobacteriaceae, especially multi-drug-resistant strains. However, the emergence of carbapenem-resistant Enterobacteriaceae has become a major public health problem worldwide leading to limited treatment alternatives resulting in increased morbidities, mortalities and healthcare costs. This prompts for a need to clarify the resistance mechanisms as well as rapid and accurate detection of resistance if effective control is to be achieved. This study aimed at determining molecular mechanisms of carbapenem resistance in Escherichia coli and Klebsiella pneumoniae that are carbapenemase negative. A total of 87 isolates (47 E. coli and 40 K. pneumoniae) were screened for antimicrobial susceptibility by disc diffusion method. The 61 meropenem non-susceptible isolates were then screened for the production of carbapenemases, Extended Spectrum Beta Lactamases and AmpC enzymes phenotypically. The 28 carbapenemase negative isolates were then screened for outer membrane porin genes, followed by sequencing to detect mutations in the outer membrane porins. Out of 87 isolates, 28 (32.2%) were resistant to meropenem and 33 (37.9%) were intermediate. Among the 61 meropenem non-susceptible isolates, 36 (59%) were Extended Spectrum Beta Lactamase producers, 8 (13.1%) were AmpC producers, 23 (41.4%) were producing carbapenemases and 28 (45.9%) were carbapenemase negative. All the 28 carbapenemase negative isolates had at least one mutation in their outer membrane porin gene sequence that either resulted into a frame shift, deletion, insertion or substitution that led to early termination and production of truncated and non-functional protein, and/or produced an extended spectrum beta lactamase or AmpC enzyme. There is a high prevalence of carbapenem resistance in Mulago National Referral Hospital. Alteration of outer membrane porin proteins associated with production of extended spectrum beta lactamases and/or AmpC enzymes could have conferred carbapenem resistance in some of E. coli and K. pneumoniae isolates. There is need for the government to sensitize the public on proper antibiotic use. Healthcare workers should also prescribe appropriate antibiotics for effective treatment of carbapenem resistant infections.
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    Phytochemical composition of Capsicum frutescens and its effect on growth, carcass, organoleptic and feed cost parameters of indigenous and broiler chickens
    ( 2022) Sebulime, Peregrine
    Chicken meat is a major source of animal protein and its production has increased globally by 25% in the last decade mostly in Asia and Africa. However, due to the ever-increasing problem of antibiotic resistance, routine non-therapeutic antimicrobial use in chicken production systems is no longer considered a reasonable and viable practice. Natural products are among the preferred replacers of antibiotics for non-therapeutic uses such as promoting growth. In addition, the chicken meat produced from systems utilizing natural products is considered safer and protective of consumer health. Herbs and spices are important sources of natural products that can be used to improve growth and carcass traits of chickens in Uganda. Capsicum frutescens is among the common spices easily accessible but its use as an enhancer of growth and carcass traits in intensive chicken production systems has not yet been investigated in Uganda. This study aimed at establishing phytochemical composition and identification of dietary inclusion levels of Capsicum frutescens powder with positive effects on growth, carcass and organoleptic parameters of indigenous chickens and on growth and carcass traits of broilers. The phytochemical composition and antioxidant activity of C. frutescens powder were determined using conventional methods. A completely randomized design was used for each of three experiments for broilers and for each of the two experiments for indigenous chickens. The three experiments on broilers were differentiated by the durations of feeding diets enriched with C. frutescens. The three durations were: 13 days, 19 days and 24 days. Each duration represented the number of days prior to 59 days of age which was the age of slaughter. In each broiler experiment, three treatments defined by three dietary inclusion levels (1.1%, 2.2%, 4.4%) of C. frutescens powder were each randomly assigned to a group of forty-five broilers housed in three pens with each pen consisting of fifteen broilers. In addition, each experiment had a control group with forty-five broilers housed in three pens with each pen consisting of fifteen broilers. The control group received only the basal finisher diet. For the indigenous chickens, one of the experiments utilised cotton seed cake as the lipid source whereas sunflower seed cake was the source of lipids in the other. In the experiment where cotton seed cake was utilised, three treatments defined by inclusion levels (1.1%, 2.2%, 4.4%) of C. frutescens powder in diets, were each randomly assigned to a group of forty-five chickens housed in three pens with each pen consisting of fifteen chickens. The control group of this experiment had forty-five chickens housed in three pens with each pen consisting of fifteen chickens. The control group received only the basal diet. The other experiment of the indigenous chickens, where sunflower seed cake was utilised as lipid source, had two replicates instead of three and the treatments as well as number of chickens per replicate were similar to that earlier described for indigenous chickens. In both experiments of indigenous chickens, the chickens were aged 199 days at the start of the experiment and the duration of the experiment was 37 days. Results indicated that C. frutescens powder has several phytochemicals including alkaloids, coumarins, saponins, anthracenosides and reducing compounds. In addition, the total phenolic content, alkaloids and saponins were 587.75 mg GAE/100g, 6.8% and 23.5%, respectively. Further, the antioxidant activity scores based on DPPH radical scavenging activity at concentrations 100, 200, 400, and 500 ug/mL were 17.08, 20.75, 29.82, and 33.31%, respectively. Among broilers, diets formulated with 2.2% inclusion level and provided for 19 days had positive effects represented by significant increase in final body weight (1257.50±26.40g, P=0.007), carcass weight (786.18±22.39g, P=0.006) and gizzard weight (33.37±1.23g, P=0.02) by 14%, 15% and 12%, respectively relative to the control. Similarly, among indigenous chickens, diets with sunflower seed cake formulated with 2.2% inclusion level had positive effects demonstrated by significant increase in heart weight (4.00±0.33g, P=0.001), liver weight (15.30±0.80, P=0.04), proventriculus weight (4.25±0.14g, P=0.004) and intestine weight (82.50±3.58g, P=0.002) by 38%, 15%, 25%, and 21% respectively. Further, the 1.1% inclusion level increased intestine weight (68.00±2.17g, P=0.0002) by 26% whereas 4.4% inclusion level increased heart weight (3.60±0.21g, P=0.04) by 24% among indigenous chickens fed diets with sunflower seed cake. In contrast, the 4.4% inclusion level significantly decreased the body weight (912.50±32.05g, P=0.01), carcass weight (592.30±23.43g, P=0.04) and intestine weight (65.83±1.94g, P=0.001) by 12%, 10%, and 17% respectively among indigenous chickens on diets with cotton seed cake. Further, the 1.1% inclusion level decreased the liver weight (13.27±0.66g, P=0.02) by 14% whereas the 2.2% inclusion level decreased the heart weight (2.97±0.18g, P=0.001) by 19% among indigenous chickens on diets with cotton seed cake. Furthermore, results showed that none of the inclusion levels of C. frutescens had an effect on texture, taste, juiciness and overall acceptability of meat from indigenous chickens. The results of this study highlight that C. frutescens has several beneficial phytochemical compounds and high antioxidant activity for improving broiler growth and carcass yield. Further, an inclusion level of 2.2% in finisher diets for 19 days is acceptable since it has capacity to improve final body weight and carcass weight of broilers by 14% and 15% respectively relative to the control. Similarly, an inclusion level of 2.2% in diets with sunflower seed cake is recommended for indigenous chicken effective 199 days of age due to potential to improve carcass yield.