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Item Embargo In-vitro and in-silico evaluation for phytochemicals and anti-diabetic potentials of Trichodesma zeylanicum ethanolic and ethyl acetate extracts(2025-09-05) Manyuwa, Manase; Tshidino-Vukeya, S. C.; Madala, N. E.; Ramabulana, A. T.Background: Trichodesma zeylanicum (Burm. f) R.Br. belongs to the Boraginaceae family. It is an annual shrub native to Australia, Africa, and Asia. This plant contains phytochemicals with antioxidant and anti-diabetic properties of interest to people with T2DM. The present study was aimed at an in-vitro and in-silico evaluation of phytochemicals and anti-diabetic potentials of ethyl acetate (EA) and ethanol extracts from the leaf, stem, and root of T. zeylanicum. Materials and methods: Trichodesma zeylanicum was collected from Maungani village near the University of Venda. Its leaf, root, and stem were washed, dried in the shade, and ground into powder. Plant part powders were weighed separately and extracted using absolute EA and ethanol. Plant extract filtrates were concentrated using the rotary evaporator. Qualitative biochemical methods, including Thin-layer chromatography (TLC), were used for phytochemical and antioxidant analyses using ascorbic acid, gallic acid, and rutin as standards. The liquid chromatography quadrupole time of flight mass spectrometry (LC-qTOF-MS) technique was used for phytochemical analyses and identifications. In-silico α-amylase, α-glucosidase, and vascular endothelial growth factor receptor 2 (VEGFR2) inhibitory potentials of selected identified phytochemicals were screened using the Protein Data Bank available α-amylase, α-glucosidase, and VEGFR2 by molecular docking methods using acarbose and sorafenib as standard inhibitors. Results: Known and unknown phytochemicals in EA extracts of all of T. zeylanicum organs were analysed and identified. In comparison to EA, it was commonly observed that ethanol showed its potential to extract more bioactive chemicals from T. zeylanicum root (59 mg), stem (58 mg) and leaf (67 mg) extracts, as both percentage yields are high. The DPPH radical scavenging activity of EA root, stem and leaf extracts exceeds that of ethanol plant extracts at low to high concentrations, with the EA leaf extract displaying superior radical scavenging activity of up to 70.259±2.029% at 40 μg/mL when compared to ascorbic acid (86.057±0.610%) [p≤0.001] at the same concentration. Although the ethanol radical scavenging activity of root, stem and leaf extracts was extremely low, the leaf extract radical scavenging activity was moderately promising. TPC in ethanol root (11.489±0.545 μg GAE/mg dried extract), stem (10.753±1.116 μg GAE/mg dried extract) and leaf (47.187±2.300 μg GAE/mg dried extract) extracts surpass the EA root (1.604±0.206 μg GAE/mg dried extract, p ≤ 0.001), stem (2.280±0.844 μg GAE/mg dried extract, p ≤ 0.001) and leaf (2.760±0.086 μg GAE/mg dried extract, p ≤ 0.0001) extracts with ethanol leaf extract depicting significantly higher amount of the phenolic content compared to those of ethanol root and stem extracts (p ≤ 0.001). Among the selected compounds docked against two enzyme markers for diabetes and one protein for wound development, ligand 1A (4-[2,6-Dihydroxy-4-(6,7,8-trihydroxy-2-naphthyl) phenoxy]-3-hydroxy-7-(3,4,5-trihydroxyphenyl) naphthalene-1,2-dione) from EA root extract of T. zeylanicum showed the highest binding affinities when docked against α-amylase (-11,0 kcal/mol), α-glucosidase (-10,2 kcal/mol), and VEGFR2 (-9,6 kcal/mol) in comparison with acarbose (positive control) for α-amylase (-7.4 kcal/mol) and α-glucosidase (-7.8 kcal/mol), and sorafenib (positive control) for VEGFR2 (-8.1 kcal/mol) as inhibitors. The molecular networking displayed closely related human pancreatic α-amylases (AMY2A and AMY2B) and intestinal α-glucosidase (GAA) participating in the metabolism of carbohydrates, together with the involved interacting enzymes leading to T2DM, whereas the kinase VEGFR2 is not in association with these enzymes, although playing a role in wound formation from diabetic patients. Multiple sequence alignment data demonstrated the α-amylase, α-glucosidase and VEGFR2 fully conserved regions (Trp, Lys, Val, Pro, Gly, Ala, Gln, and Ser), which can support their role in digesting carbohydrates and wound formation. Conclusion: Findings in this study demonstrated that the EA root extract of T. zeylanicum contains phytochemicals with anti-diabetic potentials associated with wound healing and antioxidants, suggesting that the root of this plant can serve as a source of phytochemicals with antidiabetic and wound healing properties. Cytotoxicity of ethanol and EA extracts from different organs of T. zeylanicum, molecular dynamic simulation (MD) and an in-vitro α-amylase and α-glucosidase inhibitory activity test are recommended for further research to validate and explore the findings of the molecular docking and safety and efficacy of these plant extracts.Item Embargo Molecular characterization of microbial communities in small-scale biodigesters using whole genome shotgun metagenomic approach(2025-09-05) Matamela, Tendani; Mathomu, L. M.; Matambo, T.Increasing attention has been directed towards the role of microbial communities in anaerobic digestion (AD). During AD, microorganisms degrade organic waste, such as animal excrement, through four key phases: hydrolysis, acidogenesis, acetogenesis, and methanogenesis, resulting in digestate and biogas production. Advances in metagenomic techniques, surpassing traditional culture-based methods, have enabled a deeper characterization of the microbial populations involved in this process. However, a knowledge gap remains regarding small-scale biodigesters, particularly those in rural areas. This study employs whole-genome shotgun metagenomics integrating data from metagenomic reads, contigs, and metagenome-assembled genomes (MAGs) can achieve a more comprehensive understanding of microbial taxonomy and functional capabilities in biodigesters. This study investigates the microbial communities in small-scale biodigesters, using taxonomic data from various sources to reveal the functional roles of the microorganisms. The research identified 4 superkingdoms, 115 phyla, 107 classes, 189 orders, 322 families, 738 genera, and 2046 species. Bacteria dominated ranging from 79 to 89%, whereas archaea accounted for 11 to 20% of the community. The eukaryotic microbial’s relative abundance was less than 1%. Both read-based and contig-based classifiers found Proteobacteria, Firmicutes, and Bacteroidetes to be the most abundant bacterial phyla, followed by Actinobacteria and Chloroflexi in cow dung-fed biodigesters. The archaeal community was dominated by Euryarchaeota, with Methanomicrobiales and Methanobacteriales as the predominant orders. Functional analysis reveals that most identified microorganisms contribute indirectly to methanogenesis by playing crucial roles in hydrolysis and acidogenesis through genes involved in the metabolism of organic molecules broken down during the four phases of anaerobic digestion. RAST analysis annotated 11 methanogen-related enzymes and their abundance across different samples. Future research should focus on cultivating and monitoring these identified microbes to determine optimal conditions for maximizing biogas production in laboratory settings, thus advancing environmental bioaugmentation strategies.Item Embargo Comprehensive evaluation of cow dung digestate composition for potential soil fertilization: Advanced analytical techniques(2025-09-05) Phaphana, Fhumulani Edna; Mathomu, L. M.; Madala, N. E.Cow dung digestate, a nutrient-rich byproduct of anaerobic digestion, offers a sustainable alternative to synthetic fertilizers, aligning with the United Nations Sustainable Development Goals (SDGs), particularly Goal 2 (Zero Hunger) and Goal 13 (Climate Action). This study aims to evaluate the physicochemical composition, nutrient bioavailability, and agronomic potential of CDD (Cow Dung Digestate) in South African agriculture, focusing on its impact on soil fertility, microbial activity, and environmental sustainability. A multi-faceted analytical approach was employed, incorporating pH measurement, gravimetric moisture determination, and UV-VIS spectroscopy for organic matter analysis. Advanced techniques, including FT-IR, ICP-OES, GC-MS, and UHPLC-QTOF-MS, characterized functional groups, elemental composition, volatile organic compounds, and metabolomic profiles. Multivariate data analysis provided a comprehensive assessment of nutrient interactions and biochemical complexity. Results indicate that CDD maintains a moisture content of 45.95%–48.72% and an alkaline pH range of 7.73–8.62, conditions that promote microbial proliferation and nutrient solubilization. Spectroscopic analyses confirm the retention of nitrogenous compounds, humic substances, and bioactive metabolites, contributing to both immediate and long-term soil fertility. Metabolomic profiling highlights the presence of oxaluric acid, proline, and nopaline compounds associated with stress tolerance, nitrogen metabolism, and plant-microbe interactions emphasizing the bio-stimulatory potential of digestate. Structural and elemental analyses reveal strong organic-mineral interactions that enhance soil moisture retention and nutrient stability. Compared to synthetic nitrogen fertilizers, digestates particularly UD, exhibit higher levels of potassium, calcium, magnesium, and iron. Elevated sodium and chromium concentrations in certain digestates necessitate careful application strategies to mitigate salinity and toxicity risks. Additionally, lipid-derived compounds enhance microbial activity, while volatile organic compounds suggest the need for optimized composting strategies to minimize gaseous emissions. This study establishes CDD as a scientifically validated biofertilizer that reduces reliance on synthetic fertilizers while enhancing soil health, microbial diversity, and crop productivity. However, precise application protocols must be developed to balance nutrient delivery with potential ecological risks.Item Embargo Inhibitory effects of Green synthesized Senna alexandrina silver nanoparticles (SAAgNPs) on Escherichia coli DnaK(2025-09-05) Seboya, Koketso Oscar; Mathomu, L. M.Plant extracts have garnered considerable interest in the environmentally friendly and economically viable production of silver nanoparticles (AgNPs) through their application in green synthesis. However, Plant-derived nanoparticles may potentially have unique physicochemical characteristics. This study is significant in that it contributes to the understanding of the effects of AgNPs on the functional and structural integrity of the heat shock protein DnaK. The research presents an improved synthesis process for silver nanoparticles (AgNPs) from the Senna alexandrina leaf extract. In addition to evaluating the synthesized AgNP's physicochemical characteristics, the research also examines their effect on the functional activity of E. coli DnaK. During synthesis, the color change from light yellow to dark brown was observed within 30 minutes after the addition of 3 ml of S. alexandrina leaf extract to 1 mM of silver nitrate. A surface plasmon resonance peak at 420 nm revealed by ultra-violet spectroscopy was used as additional confirmation that AgNP had formed. The synthesized SA-AgNPs were characterized using UV- VIS spectrophotometry, FTIR, and SEM analysis. Furthermore, heat shock proteins (Hsp) are cellular and stress-induced proteins. With respect to this, it is anticipated that the Hsp can also interact with AgNPs transported into cells and/or the circulation system. The present study was intended to investigate the effects of synthesized silver nanoparticles (AgNPs) using locally- sourced leaf extracts of S. alexandrina on E. coli DnaK. In this study, AgNPs were synthesized using the green synthesizing method by the reduction of silver nitrate into nano-sized silver nanoparticles (AgNPs) inactivated concurrently, which was capped with the phytochemicals of S. alexandrina . FTIR, UHPLC-qTOF-MS, investigated the functional groups present in plant extract to identify the compounds responsible for reducing silver ions. Altogether, findings from this study suggest that DnaK is highly involved in E. coli cytoprotection against AgNPs. In conclusion, both the effect of AgNPs on DnaK stability to heat stress and function in suppressing MDH aggregation were investigated. More importantly, it was also observed that AgNPs can suppress heat-induced aggregation of MDH and consequently stimulate ATPase activity of DnaK. In silico docking studies revealed promising binding affinities of specific metabolites in relation to key proteins (DnaK and its co-chaperones) involved in bacterial survival.Item Embargo Biochemical and biophysical characterization of PFF1010c, a Plasmodium falciparum heat shock protein(2025-09-05) Masoga, Thabo Rolffy; Shonhai, A.Since time immemorial malaria remains a health concern despite the extensive interventions to eliminate the disease. Plasmodium falciparum, the agent for the most lethal form of the disease, survives insults imposed by the human host response and the adverse effects of antimalarial drugs, thereby building resistance against this intervention. Amongst other means, the parasite employs the heat shock protein (HSP) machinery in response to the imposed hostilities. Compared to other malaria parasites, P. falciparum expresses a large complement of an HSP family known as the J domain proteins (JDPs). Hsp70 is a prominent molecular chaperone whose chaperone function is coordinated by JDPs. While several JDPs have been characterized, the role of PFF1010c, a JDP of P. falciparum remains unknown. This study explored the structural and functional features of PFF1010c using in silico approaches (BLASTP, PSIPRED, CLUSTALW, MEGA11, SOPMA, CDD/SPARCLE ESPRIPT, Alphafold, GalaxyHomomer, ProtParam, PlasmoDB, and STRING). A plasmid construct expressing recombinant PFF1010c was expressed using E. coli XL1 Blue. The secondary structure of the protein was investigated using circular dichroism spectroscopy. PFF1010c thermostability was assessed by ANS probe based extrinsic fluorescence spectroscopy. The functional cooperation of PFF1010c with PfHsp70-1 was investigated by monitoring refolding of heat-denatured luciferase in vitro. PFF1010c appeared to assume a fold constituted by two domains: the SVN motif-containing J domain and the Cterminal domain. PFF1010c was found to be thermostable. Biochemical and in silico studies suggested that the protein self-associates through the C-terminal domain. Also, PFF1010c abrogated the refolding activity of PfHsp70-1. Although PFF1010c appeared to interact with PfHsp70-1, it wasincapable of stimulating PfHsp70-1 chaperone function. This suggests that PFF1010c may interact with PfHsp70-1 possibly to improve the holdase chaperone function of PfHsp70-1 as well as to conserve the energy economy of the parasite cell.Item Embargo Ethinylestradiol (EE2) and Human Reproductive Health: Investigating the Potential Effects of Contraceptives on Human Reproductive Health and Sexually Transmitted Diseases in the Era of HIV and AIDS(2025-09-05) Manavhela, Murendeni; Samie, A.; Traore, N. A.Background: Reproduction and sexual health are fundamental aspects of human life and are influenced by various hormones, including sex steroids. Sex steroids, estrogen in particular have been implicated in numerous human health issues. The association between estrogens and sexually transmitted diseases (STIs) is not clear as studies are contradictory; other studies suggest that estrogens confer protection against STIs and others report that they facilitate the progression of STIs. Because of the extensive use of Ethinylestradiol (EE2) in birth control, it has now become a major global pollutant due to its presence in wastewater, either through household waste, agricultural waste or through industrial wastes. In the aquatic environments where wastewater is discharged, there is a wide variety of organisms that reside there, and in the end they become victims of the impact the hormones has have on them. It’s been reported that exposure of aquatic organisms to estrogens has been linked to endocrine disruption, including feminization, and these changes are correlated with EE2 concentrations. While extensive research has been on the ecological consequences of EE2, there remains a gap in understanding the impact of estrogens in human health, particularly synthetic estrogens. Given the widespread environmental distribution of these hormones, further research is necessary to elucidate their potential effects on human reproductive health, particularly in relation to sexually transmitted infection (STIs). Aims and Objectives: The main aim of the study was to further our understanding of the potential impacts of birth control and the tools used particularly ethinylestradiol on sexual health and sexually transmitted infections. A number of objectives were identified and included: 1. To assess the knowledge, attitude, and practices of contraceptives amongst women of reproductive age. 2. To determine the prevalence of contraceptive use amongst women of reproductive age. 3. To describe contraception association with sexually transmitted diseases. 4. To identify microorganisms causing sexually transmitted infections among study participants (Neisseria gonorrhea, Chlamydia trachomatis and Trichomonas vaginalis). 5. To determine ethinylestradiol concentration in the study population. 6. To assess the potential association between contraceptive use and UTI. 7. Provide insight into urine metabolomics across study participants using different birth control methods. Methods: A community based cross-sectional study was conducted in Northern Limpopo, in the Vhembe district. A paper-based questionnaire was used to collect data such as demographic data, knowledge, attitude and practice of women of reproductive age towards contraceptives. Urine samples were collected from the study participants using sterile sample collection containers. In the laboratory, urine samples were cultured for UTI determination. Enzyme-linked immunosorbent assay (ELISA) was used to quantify Ethinylestradiol (EE2) concentration from the samples. Polymerase Chain Reaction (PCR) was used for detection of three different microorganisms causing non-viral sexually transmitted infections including Chlamydia trachomatis, Neisseria gonorrhea and Trichomonas vaginalis. For metabolite extraction, solid phase extraction (SPE) was used to extract metabolites from urine samples using C18-E silica- based cartridges. Collected data was entered and cleaned on excel spread sheet. SPSS software V27 and Stata V12 were used to analyze the data (descriptive statistics was used to estimate frequencies and chi-square test was used for examine associations). Metabolomics data was analyzed using MetaboAnalyst 6.0 online software. Results: Of the 364 women who participated in the study, 96.11% were aware of what contraceptives are. Participants acquired knowledge through more than one platform, with 41.67% through media, 41.38% via health care workers and 31.61% through social media networks. Participants had knowledge of at least one method of contraception. In practice, 75.1% were using at least one form of contraceptive and the most common methods were injectables (46.1%), male condoms (23.8%), and pills (17.4%). Most participants (96.9%) had a positive attitude towards contraceptives and 94.4% said they think contraceptives are beneficial, while 79.8% agreed that the main benefit is to avoid unwanted pregnancy. Chlamydia trachomatis was the most common STI with a prevalence of (17.39%), followed by Neisseria gonorrhea with a prevalence of (5.43%) and Trichomonas vaginalis with a prevalence of (2.72%). Based on the age group, N. gonorrhea had the highest rate of (75.0%), followed by C. trachomatis with (59.68%) and then T. vaginalis with a prevalence rate of (30.0%) in participants who were aged between 20-30 years. Educational status appeared to have an influence on the prevalence of N. gonorrhea showing the highest infection rate of (55.0%) in individual’s who studied up to tertiary level as well as C. trachomatis (53.33%) and T. vaginalis (50.0%) in those with high school education. Coinfections with C. trachomatis and N. gonorrhea were observed in (30.0%) of the participants. Urinary tract infections (UTIs) were detected in 34.2% of the study population. Education did not seem to reduce the impact of UTIs among the study participants. UTIs were statistically significant among patients who had been using contraceptives for a long time and also among patients who were using Intrauterine devices as contraceptive method particularly (p value less than 0.05. UTIS were also common among participants who had experienced miscarriage although the difference was not statistically significant. The presence of blood, nitrate and ketones in the urine samples were significant markers of UTI among the study participants. The concentration of ethinylestradiol varied across the samples from low concentration of 11pg/ml to the highest concentration of 804 pg/ml. The concentration of EE2 varied with age, marital status and education level. Individuals with higher educational level also seemed to have higher concentration of EE2 in their urine samples. There was a statistically significant association between ethinylestradiol occurrence and urinary tract infection (UTI) with p value of 0.006. However, there was no significant association with the three STDs tested, although more samples positive for the infection tended to be positive for EE2. Individuals who had experienced miscarriage tended to have higher concentrations of EE2 although the difference was not statistically significant. Metabolomics studyies identified a number of compounds in the urine samples. The metabolites identified were mostly associated with lipid metabolism changes, hormonal and estrogenic activity, inflammation and immune responses. Metabolites with neurological impact were also detected. Some of these metabolites included but are not limited to Equol, Pergolide, Leukotriene and Scopamine. Conclusion: We observed a high rate of awareness towards family planning methods. However, there is still a need for improvement in terms of translating the information into practice. Although, women of reproductive age were the primary focus, regular screening for sexually transmitted infections (STIs) should be expanded to other significant groups such as sex workers and men who have sex with men (MSM). Ethinylestradiol (EE2) can be detected in individuals beyond those actively using EE2-containing contraceptives; however, prolonged use of such contraceptives may contribute to elevated levels of EE2 in urine samples over time.Ethinylestradiol (EE2) is not only found in individuals who are using ethinylestradiol-containing contraception, however, long term usage of contraceptive might increase the chances of increased EE2 in the urine samples. However, there is need for further studies to understand the origins of the synthetic compound as there are external factors contributing to levels of the synthetic compound in the body. Furthermore, variation of urine metabolomics is dependent on individual physiology and could be used as biomarkers for lifestyle factors, drug response and to monitor diseases.Item Embargo Indentification and molecular characterization of human papillomavirus infection among women living with and without HIV in selected health facilities in Limpopo Province, South Africa(2025-09-05) Rikhotso, Rixongile Rhenny; Bessong, Pascal Obong; Mitchell, Emma McKimBackground: Co-infection of human papillomavirus (HPV) and human immunodeficiency virus (HIV) has been well established. Both viruses are sexually transmitted with paramount public health implications due to their interaction with cervical cancer (CC). Globally, the estimated prevalence of HPV is 11.7%, with high-risk (hr-) HPV 16 and HPV 18 attributing for most cases of CC. Due to the high genetic diversity of HPV, it is important to study the virus in different geographic locations, people, and in persons living with HIV, to inform the selection of genes for vaccine improvements and potential vaccine trial sites. Consequently, it is important to describe the prevalence, genotype distribution and genomes of HPV in Limpopo Province, where there is limited data. The general objective of this study was to therefore to identify and characterize HPV DNA among women living with and without HIV in selected health facilities in Limpopo Province, South Africa. The specific objectives were: (1) To provide a narrative literature review on the prevalence and distribution of selected cervical HPV genotypes among women living with and without HIV in South Africa; (2) To determine the prevalence of cervical HPV infection among women living with and without HIV in selected health facilities in Limpopo Province, South Africa; (3) To describe HPV genotypes among women living with and without HIV in selected health facilities in Limpopo Province, South Africa; and (4) To generate and characterize HPV full genomes among women living with and without HIV in selected health facilities in Limpopo Province, South Africa. Methods: A systematic review of the available literature was conducted in the quest to understand and determine the prevalence and distribution of cervical HPV among women living with HIV (WLWH) and those without HIV in South Africa, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) workflow. Records were retrieved from PubMed and Web of Science databases. An analytic cross-sectional study on HPV prevalence and genotype distribution among 450 women who were living with and without HIV from selected health facilities in Limpopo Province was done. In this regard, total DNA was purified from cervical specimens and amplified using a double-nested polymerase chain reaction (PCR) strategy, targeting the L1 gene. A sample that shows an amplification product of 450 bp in the first nested PCR or a product of 150 bp in the second nested reaction was taken as positive for the presence of HPV DNA. Statistical analysis, using models in R-statistical package, was done to determine comparative inferences of the presence of HPV DNA and associated risk factors. HPV genotypes were determined through deep sequencing of the 450 bp PCR products on an Illumina MiniSeq platform. The generated sequences were quality assured and analyzed for viral genotypes. Rolling circle amplification (RCA) was used for enriching DNA in samples that tested positive for HPV DNA to generate and characterize near-full length HPV genomes. The enriched DNA was purified with AMPure XP beads and quantified with Qubit, followed by DNA library preparation, and then sequenced on an Illumina MiniSeq platform following the manufacturer’s protocols. Results Prevalence and distribution of selected cervical HPV genotypes in South Africa A total of sixty-nine (69) articles met the inclusion criteria for determining the prevalence and distribution of cervical HPV genotypes among South African women who were either living with HIV or not. These articles were on studies done in 5 of the 9 South African Provinces. The studies were conducted between 1989-2021. Sequencing was the least genotyping tool used in these studies, while most studies utilized PCR-based hybridization commercial kits. Compared to other investigated genotypes, HPV 16, HPV 18, and HPV 35 were predominant in the study population regardless of the HIV status. Prevalence of HPV DNA and genotype distribution in selected health facilities in Limpopo Province, South Africa The HPV DNA was detected in cervical specimens from 147 of 450 women (32.7%), significantly higher at 52.21% (p = 0.00) among WLWH compared to women living without HIV. Forty-eight samples were of acceptable quality and analyzed for viral diversity. The study detected 41 HPV genotypes, all of which belong to the Alphapapillomavirus genus of the Papillomaviridae family. In general, HPV 45 (16.7%) was the predominant genotype. However, HPV 81 (18.8%) and HPV 56 (25.0%) were the most common genotypes among women living with and without HIV, respectively. Multiple infections and high-risk HPV genotypes were more common among WLWH. Characterization of whole genomes of cervical human papillomavirus Six full genomes of HPV genotypes 34 (n=1), 35 (n=1), 42 (n=1), 45 (n=2), and 90 (n=1) are reported herein. The study further identified variant sub-lineages, including the A1 sub-lineages of hr-HPV 35 and hr-HPV 45. The genome sequences are available at the National Center for Biotechnology Information (NCBI) in the Sequence Read Archive (SRA) database. Interpretations and conclusions Prevalence and distribution of selected cervical HPV genotypes in South Africa The narrative review of the literature indicated that there are limited studies on cervical HPV prevalence and genotype distribution in South Africa. Therefore, further research is required to identify HPV genotypes, including the use of sensitive approaches such as next generation sequencing. This will enhance our understanding of HPV geo-diversity patterns in various provinces across South Africa, and subsequently inform decisions on screening strategies, and selection of geographically relevant genes for vaccine improvement and development. The high prevalence of hr-HPV 16, HPV 18 and HPV 35 regardless of HIV status in the country is a public health concern, considering their significant contribution to the burden of CC. Consequently, routine HPV screening and prevention measures such as HPV vaccination for all individuals remain critical. Because current vaccines exclude HPV 35 as a target, there is a need to consider this genotype in future vaccine development efforts. Prevalence of HPV DNA and genotype distribution in selected health facilities in Limpopo Province, South Africa The study reported a relatively high prevalence of HPV infection among the study participants, which signified the burden of HPV in the province. The prevalence was significantly higher among WLWH, including more HPV genotypes and hr-HPV genotypes as compared to women living without HIV, potentially due to immunosuppression. Considering the high rate of HPV-HIV co-infection observed in this study and other studies in South Africa, prioritizing women living with HIV for cervical HPV screening and HPV vaccination is recommended. Strengthening integrated HPV and HIV screening programs and ensuring regular monitoring of cervical health among WLWH remains a public health imperative. Moreover, the study also showed that women living with and without HIV may have different profiling in terms of HPV infections which is important in intervention strategies, example development of targeted HPV screening strategies. The current study also fills a significant gap of limited data by providing baseline information on the prevalence and genotype distribution of cervical HPV infection among women living with and without HIV in the study area. The study findings further encourage national-level surveillance with the inclusion of expanded regional representation. Characterization of whole genomes of cervical HPV Near full-length genomes of six HPV genotypes were successfully characterized in the study population. These sequences could serve as a valuable resource for further research and vaccine development studies. The identified variant sub-lineages may underscore the need to strengthen HPV vaccine coverage, routine cervical screening, and genomic monitoring in underrepresented areas such as Limpopo Province. Moreover, this data supports the development of risk stratification models aimed at identifying women at high-risk for persistent infection and progression to CC, thereby enabling early, tailored interventions to reduce the HPV related disease burden.Item Open Access Identification and characterization of enterobacter species causing decay of onions and their antimicrobial susceptibility(2025-09-05) Lukheli, Elelwani; Traore, A. N.; Potgieter, N.Background: Plants are susceptible to a range of health problems, and onions are no exception. While onions are relatively resilient, they are not immune to diseases and pathogens. Onions that are packed before being fully dried are prone to rapid decay. Research has shown that water can facilitate the transmission of pathogens and contribute to microbial contamination of fresh produce, including Salmonella and E. coli. Salmonella can enter the soil through agricultural practices such as pesticide application, the use of fertilizers derived from animal manure, and irrigation with contaminated water. Since irrigation water can come from various sources—such as municipal supplies, treated wastewater, rivers, or groundwater—farmers are advised to safeguard their water sources to reduce the risk of contamination. Objective: To identify and characterize Enterobacter species causing decay of bulb onions and their antimicrobial susceptibility. Methods: This study was carried out in the Vhembe district in the Limpopo province. The focus was on farms in various municipalities, including both commercial and subsistence farms, as well as the rivers surrounding these farms. Water, soil, and onion samples were collected. The presence of total coliform and E. coli was determined using the Colilert Quanti tray method. Collected samples were further analyzed for Enterobacteriaceae using membrane filtration and culture-based methods. Biochemical tests (Vitek-2-system) were used to identify and confirm the isolates. E. coli pathotypes were characterized using multiplex PCR. The characterization of strains from Enterobacter species was done using Sanger Sequencing. Results: Measured temperatures ranged from 19.8°C to 25°C, with Farm 2 showing slightly elevated values. Electrical conductivity (EC) values were within acceptable limits (<540 μS/cm) across all farms, though Farm 3 exhibited higher total dissolved solids (TDS) levels, reaching up to 450 mg/L. The pH values across farms ranged from 5.0 to 6.4, lower than the recommended 6.5–8.5 for agricultural water. Elevated TDS and acidic pH in Farm 3 was observed. High levels of total coliforms (up to 2419.6 MPN/100 mL) and the presence of E. coli were detected in water and onion samples, particularly from Farms 2 and 3. PCR analysis identified multiple E. coli pathotypes, including EPEC, ETEC, EAEC, and EIEC, with greater diversity in Farms 2 and 3. Membrane filtration and culture methods confirmed the presence of gram-negative bacteria (GNB) in most samples. Pathogens such as Enterobacter cloacae, Klebsiella oxytoca, and Pseudomonas aeruginosa were isolated and confirmed with the Vitek 2 system. Enterobacter cloacae was consistently detected in onion samples, Antimicrobial Resistance: Antibiotic susceptibility testing revealed multidrug resistance among Enterobacter cloacae, Klebsiella oxytoca, and Pseudomonas aeruginosa. Resistance to ampicillin, colistin was noted. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter Ludwigii. These findings highlight the potential risks of waterborne pathogen transmission and the need for stringent water treatment and management practices to safeguard agricultural and public health.Item Embargo Characterization of the structure-function features of the R2TP complex of Plasmodium falciparum(2025-09-05) Luthuli, Sifiso Duncan; Shonhai, A.; Houry, W.Malaria remains a major cause of death worldwide. Plasmodium falciparum is responsible for the most severe form of malaria. The malaria parasite life cycle involves stages of development that span across the cold-blooded mosquito vector and a warm-blooded human host. Maintaining protein homeostasis across these physiologically distinct life stages of the parasite is mainly accomplished by a family of proteins termed molecular chaperones that assist in protein folding. Heat shock protein 90 (Hsp90) a molecular chaperone of P. falciparum is known to be important for cell development and signal transduction. The protein clientele of this chaperone runs in several hundred and efforts are ongoing to account for all its cellular clients. R2TP complex acts as a chaperone or the assembly of critical complexes in the cell, hence it is implicated in the assembly of the Hsp90 functional complex. Hence, this study aims to characterize the structural-functional features of the R2TP complex of P. falciparum. Bioinformatics analysis has revealed unique features of this complex with 3 RUVBL genes as opposed to its orthologues from yeast and humans whom both have 2 Rvb and 2 RUVBL genes, respectively. The study involved the design of antibodies and biophysical characterization of PfRUVBL proteins and their functional features. Biophysical characterization of PfRUVBL1, PfRUVBL2, and yeast Rvb1 was conducted using tryptophan spectrophotometry and limited proteolysis. In addition, surface plasmon resonance (SPR) was used to explore the oligomerization of the proteins. The ATPase activities of PfRUVBL1 and PfRUVBL2 were further investigated. Furthermore, the holdase chaperone activities of the proteins were investigated by monitoring the capability of the proteins to suppress heat-induced aggregation of a model protein, malate dehydrogenase (MDH). P. falciparum recombinant proteins showed abilities to bind nucleotides and revealed ATPase activity. In addition, these proteins revealed capabilities to self and hetero associates as suggested by SPR and slot blot assays.Item Embargo Isolation, identification, and characterization of antibiotic-producing microorganisms from soil(2025-09-05) Khwathisi, Adivhaho; Samie, A.; Madala, N. E.; Traore, A. N.The emergence and spread of antibiotic resistance in pathogenic bacteria have posed a serious concern in global healthcare. Highlighting a need for novel antimicrobial agents with diverse mechanisms of action to combat current pathogenic threats. Soil-borne bacteria are constantly exposed to various environmental stresses, which leads to the production of antimicrobial compounds as a strategy for their survival. In this context, soil-borne bacteria are key producers of antimicrobials with valuable applications in medicine, agriculture, and animal husbandry. The main aim of this study was to isolate, identify, and characterize microorganisms with the potential to produce antimicrobial compounds from soil. Preliminary screening was conducted using agar well diffusion, revealing that the bacterial isolates exhibited significant antimicrobial activity against the four pathogenic bacterial strains tested (Chapter 3). Furthermore, the use of 16S rRNA sequencing aided the identification of the active bacterial isolates at the molecular level, where isolates 1 and 2 were identified as strains of Bacillus pumilus, whilst isolate 3 was found to be Bacillus subtilis (Chapter 3). This study further explored the chemical composition of the bacterial extract using ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and classical molecular networking. Computational metabolomic analysis revealed that metabolites from these soilborne Bacillus species are predominantly composed of peptides, dipeptides, benzenoids, organic acids, and derivatives. Interestingly, the methanolic crude extract of the Bacillus subtilis isolate was mainly characterized by the abundance of dipeptides and lipopeptides, such as surfactins, suggesting that the combination of cyclopeptides and lipopeptides together could be the attribute of the enhanced antimicrobial activity observed in agar well diffusion (Chapter 4). The current study further explored the applications of Whole Genome Sequencing (WGS) to enhance understanding of the overall distribution of genomic elements that contribute toward the adaptability and biosynthesis of antimicrobial compounds (Chapter 5). The results of the WGS showed that Bacillus species presented several genes for adaptability, such as genes that encode for motility, quorum sensing, stress response, desiccation tolerance, heavy metal tolerance, synthesis of siderophores, and most genes involved in antibiotic biosynthesis, such as NRPS, beta lactone, RiPP, and terpene gene clusters. Furthermore, the use of WGS aided a more precise taxonomic classification of the identities of the strains, highlighting the importance of WGS in the identification of bacterial isolates. Lastly, inspired by the diverse chemical composition and bioactivity of Bacillus subtilis, this study further explored the potential to enhance metabolite detection and expand the data coverage in the data-dependent acquisition (DDA) mode of the UHPLC-qTOF-MS technique (Chapter 6). Interestingly, using Central composite design (CCD), this study has demonstrated that even though both collision energy and intensity threshold independently enhance metabolite coverage, it is noteworthy that when both parameters are combined, the comprehensiveness in maximizing metabolome coverage is even greater. Therefore, revealing that to enhance exploration and maximize the potential of untargeted metabolomics, this study recommends future studies transitioning from single-point optimization to group-based optimization. Overall, contributes to the growing field of natural product discovery and provides insights into the biosynthetic pathways and insight into the chemical diversity of microbial secondary metabolites. Furthermore, these findings highlight the diverse potential of microbial-derived compounds as potential sources of new bioactive molecules.Item Embargo The production and gene polymorphisms of tumor necrosis factor-alpha in patients with tuberculosis in the Vhembe District, Limpopo Province(2025-05-16) Molepo, Mmasehlare Stellah; Traore, A. N.; Magwalivha, M.; Potgieter, N.Background: Tuberculosis (TB) is an old, irresistible infection caused by Mycobacterium tuberculosis (Mtb). The causative agent has antigens that can stimulate the production of cytokines via the mononuclear phagocyte system. Although mutations in immune-related genes may directly impact a host’s ability to control the infection when exposed to M.tb, the pro-inflammatory cytokine Tumor Necrosis Factor- α is crucial in host defence against TB and granuloma formation. Therefore, this study aimed to assess the production and gene polymorphisms of TNF-α in patients with TB in the Vhembe district, Limpopo province. Methods: This study recruited thirteen TB patients from three healthcare facilities in the Vhembe district, Limpopo province. Collected samples (sputum) were analysed using the Allplex/Anyplex kit, to detect the presence of Mycobacterium tuberculosis. Serum was collected from the blood and used to determine the levels of TNF-α in the participants, using the DIAsource ELISA kit. Genomic DNA was extracted from blood samples and purified using the Zymo kit from Inqaba Biotec. The purified DNA was quantified using the Nanodrop 8 from Thermo Fischer. The quantified DNA was analyzed by MassARRAY system to sequence various TNF-α SNPs. Data analysis for this study was carried out using the Jamovi software for windows version 2.5.3. Results: This study found that about 69% of the participants were male and 62% were ≤48 years of age. The majority of the participants (62%) were unemployed. Additionally, 46% of the sputum samples were positive for Mtb. There was a high prevalence (69%) in participants with moderate TNF-α levels. Furthermore, this study found ten SNPs namely: rs10242595, rs1524107, rs1799724, rs1799964, rs1800629, rs1800630, rs1800750, rs3093662, rs309376, and rs361525. The following alleles were reported to have higher frequency than the other: rs10242595 A* (58%), rs1524107 C* (58%), rs1799724 C* (100%), rs1799964 T* (93), rs1800629 G* (88), rs1800630 C* (92), rs1800750 G* (85), rs3093662 A* (73%) and rs361525 A* (100). The allele frequency in rs309376 was evenly distributed G* (50%) and A* (50%). Conclusion: Our findings suggest that there’s a correlation between TNF-α levels and risk factors and also, there’s a significant association between TNF-α SNPs and TNF-α expression levels.Item Embargo Detection of antibiotic resistant Escherichia Coli in children living in a rural community of Lwamondo Village in Limpopo Provice and their environment(2025-05-16) Mphego, Mpho; Ledwaba, S. E.; Potgieter, N.Background: Escherichia coli (E. coli) is found almost in every environment and in the human body. Human-animal-environment interactions may be driving the spread of antibiotic resistant bacteria, particularly in areas with little restrictions on antibiotic use, widespread food animal production, and free-roaming domestic animals. Children who are exposed to domestic animals and their waste in the home environment are more likely to have intestinal colonisation of antibiotic-resistant bacteria. The main aim of this study was to detect the antibiotic resistance patterns of E. coli isolates from children less than 5 years old and from their environment. Methods: In this study, a total of 94 samples were collected from children (47 stool samples) and the environment (47 soil samples, in each household where the stool was obtained). Isolation of E. coli was done using standard culture methods on Eosin methylene blue and MacConkey agars. The Kirby Bauer disk diffusion method was used to determine the antibiotic resistance of E. coli isolates. The DNA of all E. coli isolates was extracted using the boiling method following standard protocols and confirmation of the presence of E. coli and determination of resistant genes was done using PCR. The E. coli isolates were then further tested for phylogenetic grouping using PCR employing three specific genes, namely TspE4c2, yjaA.1 and chuA. Results: The results showed that 216 isolates (117 from stools and 99 from soil samples) were presumptively identified as E. coli. A total of 211 isolates were confirmed as E. coli using PCR. Antibiotic resistance testing showed high resistance to chloramphenicol in soil (50%) and stool samples (41%). A total of 33% (70/94) isolates were positive for blaTEM gene in both soil and stool samples. Phylogroup A was predominant [87%; 65/75] followed by phylogroup D [13%; 10/75], and phylogroup B [1%; 1/75]. Conclusion: The soil environment plays an important role in the transmission of antibiotic resistant E. coli in young children. It is critical to emphasize the need of adhering to proper hygiene standards and the appropriate use of antibiotics.Item Embargo Genotyping antibiotic resistance properties of escherichia coli and campylobacter jejuni associated with diarrhoea in young children in the Vhembe District(2025-05-16) Karambwe, Simbarashe; Potgieter, Natasha; Traore, Afsatou NdamaBackground: Diarrhoea continues to threaten the lives of young children in Sub-Saharan Africa. While antibiotic resistance among enteric pathogens such as Escherichia coli (E. coli) and Campylobacter spp. is increasing, surveillance of antimicrobial resistance genes (ARG) is limited in Africa. The need for such studies in Africa was demonstrated by our published review of the literature on surveillance of molecular resistance mechanisms like blaCTX-M. Therefore, this study sought to investigate the genotypic (blaCTX-M and gyrA) antibiotic resistance profiles of bacteria causing diarrhoea in young children in the Vhembe district. Methods: A cross-sectional surveillance was done between August 2020 and August 2021. Diarrhoeal (lose, watery) and non-diarrhoeal (normal, solid) stool samples were collected from children under the age of five at selected hospitals and clinics around the Vhembe District. The Kirby Bauer Disk Diffusion technique was used to screen for antibiotic susceptibility, and PCR and sequencing were used for molecular characterization of antibiotic resistance genes. Results: Of the E. coli positive samples, 39% (18/46; 12 diarrhoeal and 6 non-diarrhoeal) had multi-drug resistance (MDR) to at least three antibiotics, with 33% (6/18) and 11% (2/18) having fluoroquinolone (gyrA) and ß-lactam (blaCTX-M) resistance mechanisms, respectively. Five percent (1/18) of the samples carried both gyrA and blaCTX-M genes. The prevalence of Campylobacter in diarrhoeal stools was 13.8% and gyrA gene was partially detected. Conclusion: Children under the age of two in the Vhembe District continue to be at risk from diarrhoea due to antibiotic resistant Escherichia coli and Campylobacter. This study raises awareness of the prevalence of MDR, and aids medical professionals in implementing the appropriate treatment. Future research should consider concurrent studies on clinical and environmental samples to determine the possible role of livestock and river water as carriers of antibiotic resistance genes.Item Embargo Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa(2025-05-16) Tambe, Lisa Arrah Mbang; Mavhandu-Ramarumo, Lufuno Grace; Tebit, Denis Manga; Bessong, Pascal ObongBackground: The last three decades have been characterized by the re-emergence of the Coronaviridae family into the human population, causing severe respiratory disease with increased morbidity rates. A dearth of information exists on human coronavirus (HCoV) molecular epidemiology, and circulation in different populations in Africa. As the COVID-19 pandemic progressed across the globe, wastewater-based epidemiology (WBE) was proposed as an alternative tool for assessing and monitoring the occurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the community level. Additionally, through wastewater-based genomic surveillance of SARS-CoV-2, the evolutionary patterns and distribution of viral types at the population level can be comprehensively characterized. This study systematically reviewed literature published prior to the SARS-CoV-2 outbreak to investigate the prevalence and molecular epidemiology of HCoVs circulating in Africa. Secondly, this study established a wastewater-based surveillance (WBS) system to track the trends of SARS-CoV-2, investigate SARS-CoV-2 variants of concern (VOC) circulating in the population, and determine the prevalence of people infected in the Vhembe and Mopani districts. Thirdly, through WBS, to describe the molecular epidemiology of SARS-CoV-2 and document the respiratory viruses occurring in the Vhembe and Mopani districts. Methodology: A systematic literature review was conducted according to the PRISMA guidelines, to understand the prevalence and molecular epidemiology of HCoVs in Africa. For the second and third objectives, wastewater influents from seven wastewater treatment plants (WWTPs) and one waste sedimentation pond (WSP) were collected weekly from January 2021 to June 2022. Out of a total of 487 samples collected, about 75% (365/487) were positive for SARS-CoV-2 RNA by qRT-PCR. Of these, 80 met the criteria for allele-specific genotyping (ASG). Positive SARS-CoV-2 RNA detected throughout the surveillance period were compared to 7-day moving average (7D-MA) of clinical cases reported per sub-district. Next, SARS-CoV-2 RNA detected during the surveillance period was normalized using the flow rate and population size. The Spearman’s correlation coefficient was used to determine the relationship between non-normalized, and normalized SARS-CoV-2 RNA data when compared to the reported clinical cases. Finally, positive SARS-CoV-2 RNA copies with a standard deviation of less than one (SD <1) were used to predict the prevalence of people infected in the study sites using the Monte Carlo simulation model. This predicted prevalence was also compared to the 7D-MA clinical cases reported per sub-district, and the correlation between them was determined. Subsequently, samples positive for SARS-CoV-2 were subjected to whole genome sequences (WGS) using the ATOPlex next-generation sequencing method and analyzed for lineage and clade assignment using the Pangolin and Nextclade tool. Relatedness of identified sequences was determined by phylogenetic analysis. VOC was analyzed for prevalence and geographical distribution. Concordance for VOC between ASG and WGS analyses was determined. Results: Findings from the systematic literature review showed that thirteen out of 54 (24%) African countries had published data on HCoV prevalence and/or genomic epidemiology, from hospitals, clinics, homes, community gatherings, farms, and individuals at airports. The first published data on HCoV was from South Africa in 2008. There was heterogeneity in the type of tests used in determining HCoV prevalence. Two studies reported that risk factors for HCoV include exposure to infected animals or humans. The second objective, establishing a wastewater-based surveillance system was achieved. Briefly, SARS-CoV-2 viral load was detected in wastewater one week prior to increased infection cases reported at the district level during the third and fourth waves, thus serving as an early warning system. Of interest, towards the end of the surveillance period, increased SARS-CoV-2 viral load detected in wastewater were not reflected in the reported clinical cases. Comparing the reported number of cases per district to the predicted prevalence revealed more cases in the Vhembe District than in the Mopani District. Third, SARS-CoV-2 molecular epidemiology and the distribution of respiratory virus were described. A total of 60 SARS-CoV-2 full genomes were analyzed. Delta and Omicron variants were detected as early as January and February 2021, respectively, while the Beta variant was detected in July 2021. Delta variant was significantly predominant at a prevalence of 45%, followed by Omicron (32%), and Beta (5%). Eighteen percent (11/60) of the sequences were assigned a lineage by Pangolin tool, but not a specific WHO variant name. Upon phylogenetic analysis, some of these sequences were seen clustering with the Alpha (2/11) and Delta (2/11) variants, while the remaining sequences clustered with each other. Mutations in the receptor-binding domain (RBD) of the Spike protein (S-protein) were investigated, with some peculiarities observed such as mutation E484K absent in all Beta variant study sequences. Three previously undescribed mutations (A631S, V327I, D427Y) were detected in Delta variant sequences. Concordance in variant assignments between allele-specific genotyping and WGS was seen in 51.2% of the study sequences. Respiratory virus surveillance revealed year-round circulation of human Adenoviruses (HAdVs), while HCoVs, influenza viruses and human parainfluenza viruses (HPIVs) were mostly detected in winter. Influenza A and B viruses (IAV and IBV) detected in the study site in 2021 were remarkably different from those reported in circulation nationwide by the NICD. Specifically, IAV (H5N1)/Guandong, a highly pathogenic influenza virus, was detected, although at a low frequency. Discussion and Conclusion: The systematic review revealed that despite the outbreaks of SARS in Southeast Asia in 2002 and MERS in 2012 in the Middle East, the quantum of virologic investigations on HCoV on the African continent was scanty. Pandemic preparedness requires cognizance of disease outbreaks in other continents, establishment of test and surveillance protocols, and infrastructure for eventualities. Regarding the establishment of a wastewater-based surveillance system for SARS-CoV-2 monitoring, this study demonstrates effective surveillance over an extended period in rural settings. This is important because most reports about the application of WBE for monitoring SARS-CoV-2 and circulating variants are predominantly from more urbanized regions in South Africa and other parts of the world. Thus, it reveals applicability of monitoring pathogens in rural areas, despite challenges encountered such as poor or non-existent sewerage systems. Such challenges are common in the African continent, highlighting the need for more of such investigations to strengthen pandemic preparedness measures. The presence of Delta and Omicron VOCs observed prior to other reports in South Africa highlights the importance of population-based approaches in genomic surveillance over approaches that rely on individual samples. Again, it also emphasizes the need for pandemic preparedness efforts to be extended to all geographic regions. Wastewater is known to potentially capture more viral diversity, including SARS-CoV-2 genetic diversity, and could reveal new viruses and VOCs in circulation before they emerge in the wider human population. Thus, continuous surveillance is necessary for documentation of cryptic lineages, which may contribute towards improving vaccine.Item Open Access Genetic diversity of Human Herpesvirus Type 8 in Northern South Africa(2024-09-06) Raphalalani, Mulalo; Bessong, Pascal Obong; Mavhandu-Ramarumo, Lufuno GraeBackground: Human herpesvirus type 8 (HHV-8), is an oncogenic virus responsible for causing all forms of Kaposi`s sarcoma (KS). HHV-8 prevalence varies globally, however, it is more prevalent in African countries, with South Africa having over 50% of HHV-8 infections. HHV-8 encodes a highly diverse open reading frame (ORF) K1 gene, which has led to the identification of seven major genotypes (A-F and Z) that are heterogeneously distributed across the world. The viral genetic landscape of any geographical area is of paramount importance in vaccine development and diagnostics. However, data on HHV-8 genotypes is scarce in northern South Africa. Therefore, this study will provide genetic diversity of HHV-8 in northern South Africa, and this may aid in the selection of genes for vaccine development. Objective: The main objective of the study was to describe the genetic diversity of human herpesvirus type 8 in northern South Africa. Methodology: Deoxyribonucleic acid (DNA) was extracted from 115 archived mouthwash samples collected from five healthcare facilities in northern South Africa. The partial open reading frame (ORF) K1 gene (~840bp) was amplified in a two round conventional PCR using JumpStart REDTaq master mix. The band of interest was extracted by phenol-freeze protocol and enriched using conventional PCR. Enriched amplicons were purified and sequenced in an Illumina MiniSeq platform. K1 genotypes were inferred using an online BioAfrica HHV-8 subtyping tool and confirmed by computing a phylogenetic tree. Intra-genetic diversity among HHV-8 genotypes was described by aligning study sequences with their respective prototype strains. Synonymous and nonsynonymous mutation rates were computed by the online SNAP tool. Results: K1 gene was successfully amplified in 61.7% (71/115) samples, along with unspecified DNA bands. The band of interest was successfully recovered in 67 amplicons (94.4%). Sixty-five gel extracted products (65/67; 97%) were successfully enriched and purified using magnetic beads. Of the 65 purified samples, 63 were sequenced using Illumina MiniSeq platform. Thirty-seven sequences had an acceptable nucleotide base call. The prevalence of HHV-8 in the study sequences was 94% (35/37) and majority of the sequences (24/35;68%) had sequence reads that span partial or complete K1 gene. Two major genotypes were detected (A and B); genotype B (19/24;79%) had a higher prevalence than genotype A (5/24; 21%). All sequences which grouped with genotype A were further classified as subtype A5. Interestingly, all sequences that were classified as genotype B did not cluster to any of the B subtypes. A higher genetic drift was observed among the study sequences reaching up to 33.7% at the amino acid level. Genotypes A and B exhibited 16.67% and 7.41% intra-genetic diversity at the amino acid level, respectively. Several amino acid polymorphisms were observed at the ITAM region of genotype A sequences (OUHC 013 and ODF 029), while the ITAM region of the B sequence was conserved. Conclusion: In this study, a predominance of HHV-8 genotype B was observed in northern South Africa. Additionally, there was a high degree of evolutionary divergence among the studied sequences. A higher frequency of nonsynonymous mutations was detected at the ITAM region of A5 sequences and these mutations may potentially affect the functionality of ITAM.Item Open Access Prevalence of Injectable Anti-Tuberculosis Drugs and Drug Resistance Amongst TB Patients in The Vhembe District (Limpopo, South Africa(2024-09-06) Patel, Sana Mustakahmed; Traore, A. N.; Magwalivha, M.; Potgieter, N.Background: Tuberculosis is one of the most infectious diseases and a major contributor to high mortality and morbidity rates worldwide. The rise of human immunodeficiency virus cases also compounds to the tuberculosis burden, as human immunodeficiency virus patients are thought to be more susceptible to tuberculosis, since the disease is an opportunistic infection and makes an individual’s immune system less capable of fighting off infections. Drug resistance tuberculosis makes transmission and combatting of disease challenging, particularly multi-drug resistant strains that are resistant to at least isoniazid and rifampicin, pre-XDR, resistant to isoniazid, rifampicin and a fluoroquinolone and extensively-drug resistant, a rare form of multidrug resistance tuberculosis that is resistant to isoniazid, rifampicin, fluoroquinolone, and a secondline injectable (amikacin, capreomycin, and kanamycin). Resistance to fluoroquinolone and injectable drugs is not frequently tested due to budget constraints and the ongoing high tuberculosis burden in developing nations. Furthermore, there is insufficient knowledge about extensively-drug resistant strains and mutations connected to fluoroquinolone and injectable drugs in South Africa. Thus, the purpose of this study was to determine the frequency of genetic mutations in M. tuberculosis obtained from tuberculosis positive patients attending medical facilities in the Vhembe region of the province of Limpopo, South Africa, with respect to genotype conferring resistance for INH, RIF, EMB, PZA, FQs, all of which are linked to resistance to second-line drugs. Methods: Ethical clearance was obtained, a total of 50 morning sputum samples were collected from health-care facilities. Structured questionaries were also administered to collect additional information. Collected data was analysed to investigate the risk factors associated with drug resistance tuberculosis. Allplex Multiplex polymerase chain reaction permitted simultaneous amplification and detection of target sequence of Mycobacterium tuberculosis. In order to determine genetic mutations, 8 single-nucleotide polymorphisms were targeted using MassARRAY (Agena) system. Results: Among the 50 tuberculosis recruited patients (from the pool of main study August 2022- June 2023), 60% were males and 40% were females. HIV was highly (66%) prevalent amongst study population; susceptible tuberculosis 4%, MTB & NTM 28% (co-infection), NTM 58% and 4% drug resistance were observed. Overall, of the SNPs designed only 8 related gene mutations could be successfully detected. Overall, from the study population, 2 samples were detected to be pre-XDR (FQ-R and RIF-R + INH-R + FQ-R). Conclusion: The findings of the study provide valuable insights into the characteristics and prevalence of tuberculosis, along with associated risk factors and co-morbidities among the study population in Vhembe district. The emergence of drug resistance highlights the necessity for continued pattern monitoring and the application of suitable treatment approaches in order to stop the spread of drug-resistant strains. To draw firm conclusions about drug-resistant tuberculosis risk factors in patients in the Vhembe region, further information is still required.Item Open Access Prevalence of multi-drug resistance tuberculosis MDR-TB and associated risk factors among patients in Vhembe Region of Limpopo. South Africa(2024-09-06) Mahamud, H.; Traore, A. N.; Potgieter, N.; Rikhotso, M. C.BACKGROUND: The increasing prevalence of drug-resistant tuberculosis (DR-TB) poses a critical and significant obstacle in the management of tuberculosis (TB) worldwide. This driven by the emergence of multidrug-resistant tuberculosis (MDR-TB) strains and co-infection of TB with human immunodeficiency virus (HIV) have become major challenge in eradicating TB especially in low- and middle-income countries. Approximately half a million cases of rifampicin-resistant tuberculosis were reported in 2020, with 78% of these cases developing into MDR-TB. AIM: This study aimed to investigate the prevalence of MDR-TB and its associated risk factors in Vhembe region (Limpopo, South Africa). METHODOLOGY: A structured questionnaire was used to collect data on plausible risk factors. A total of 50 participants from the overall study were enrolled and sputum samples were obtained from patients receiving treatment from 20 healthcare facilities. Each patient blood sample was first tested for HIV using rapid test. For this study DNA was extracted from the sputum samples using reagents from multiplex real-time PCR kit (Allplex). Multiplex realtime PCR (Anyplex/ Allplex) was used to confirm the presence of TB and to detect MDR-TB respectively. MassArray was used to detect any mutation on the SNPs for rpoB and katG and inhA promoter region of the MTB. RESULTS: Out of the 50 TB patients, 18% (9/50) were found to be MTB, 18% (9/50) to be MTB+NTM and (27/50) were NTM and 10% (5/50) could not be amplified. In this study, it was found that 2 samples were DR-TB from the patients that were MTB and MTB+NTM positive. Overall, the percentage of DR-TB was 4% (2/50). HIV prevalence in the population was 64% (32/50). The risk factors that showed high significance level of association are educational level (52%), occupation (60%), religion (80%), dusty area (72%), wearing protective mask (72%) and family support (90%). Sequence analysis showed no mutation in one patient and for the other patient the DNA not successfully genotyped. Conclusion: There are several risk factors that are associated with TB, this includes education level, occupation, and religion. High prevalence of TB (32%) was found. However, there was low prevalence (4%) of DR-TB found (Rifampicin resistant, isoniazid resistant). Furthermore, after genotyping the resistant SNPs using MassArray, the results obtained showed that there was no mutation identified. It was observed that adolescents and young adults are more susceptible to acquiring TB and DR-TB. Timely detection of medication resistance is imperative for the efficient management of the disease and dissemination of information related to TB and MDR-TB is vital especially in low- and middle-income countries.Item Open Access Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1(2024-09-06) Ncube, Hlomphani Rachel; Shonhai, A.Plasmodium falciparum causes the most lethal form of malaria infection. Identification and development of novel antimalarial drugs is limited by the shortage of simple, cost-effective screening assays. Heat shock proteins (Hsps) are a special class of molecular chaperones essential for parasite survival and development within the human host. Hsps are also implicated in drug resistance. Hsp70 forms an integral part of the molecular chaperone system, participating in essential processes such as protein refolding, preventing aggregation and aiding degradation of misfolded proteins. The molecular chaperone PfHsp70-1 is essential for cell survival during stress and it is expressed throughout all the blood stages of the parasite. Thus, Hsp70(s) are essential for functionality in the cell and their function has been demonstrated using the complementation assay. The E. coli dnaK756 mutant strain can grow between 30- 37 ℃ but its viability diminishes at temperatures >40 ℃. In addition, the strain produces non-functional Hsp70. As such, this strain is suitable for studying Hsp70 function using the complementation assay. A high-throughput complementation assay was optimized and developed to screen potential inhibitors of Hsp70. Cells heterogously expressing DnaK were the positive control, while those expressing KPf-V436F were the negative control and the cells heterologously expressing KPf were the test cells. KPf contains a substrate binding domain (SBD) from PfHsp70-1. Protein expression was confirmed by sodium dodecyl sulfate-polyacyrlyamide and Western blot analysis. Cells grown with DMSO managed to grow normally under stress, showing that DnaK and KPf conferred cytoprotection to E. coli dnaK756 cells. Those heterologously expressing the negative control could not withstand the thermal stress. However, treatment with inhibitors colistin sulfate and pifithrin μ hindered the growth of cells heterologously expressing DnaK and KPf under non-permissive temperatures by 50-fold. Interestingly, the inhibitors seemed not to have any effect on the cells heterologously expressing the mutant protein KPf-V436F. The cells grown with DMSO seemed to die at the same rate as those treated with inhibitors. The colistin sulfate IC50 values were 0.22 μM for DnaK and 0.07 μM for KPf, whilst pifithrin μ IC50 values were found to be 14.48 μM and 10.41 μM for DnaK and KPf respectively. Statical analysis using Two-way ANOVA utilizing multiple comparison analysis proved the validity of this assay. Solubility studies were conducted to check the solubility status of E. coli dnaK756 proteome in the presence of the inhibitors. Findings from this study show that the inhibition interrupted the interaction of DnaK/KPf with some of its client proteins consequently leading to aggregation. This was a confirmation of the compromised function of DnaK/KPf due to the presence of the inhibitor. Tryptophan fluorescence studies were done to validate the findings of the complementation assay, the results show that pifithrin μ and colistin sulfate perturb the tertiary conformation of DnaK, KPf and PfHsp70-1. Since a protein’s tertiary conformation is crucial for its function, perturbations in its conformation might have altered the protein functional activity. These findings further validated the complementation assay results which show cell growth defects in the presence of inhibitors. Hence, the current study demonstrated the potential of using the complementation assay as a screening assay to identify potential inhibitors of P. falciparum Hsp70-1.Item Open Access Prevalence of drug-resistant tuberculosis and associated risk factors among patients in the Northern Region of South Africa(2024-09-06) Mphaphuli, Avheani Marry; Rikhotso, M. C.; Traore, A. N.; Potgieter, N.Background: Drug-resistant TB is a major global health threat, driving the ongoing TB epidemic and increasing morbidity and mortality worldwide. Currently, national tuberculosis (TB) control programmes are challenged by the emergence of drug resistance to more anti-TB drugs resulting in multi-drug resistant TB (MDR-TB), pre-extensively drug-resistant TB (pre-XDR-TB) and XDR-TB. Globally, about 465,000 people have rifampicin-resistant TB (RR/TB; 78% MDR-TB), where new cases contribute about 3.5%, and 18% among previously treated cases. Variety of factors, including demographic, behavioural, clinical, and environmental factors can favour the spread of DR-TB in the community. However, the major mechanism of acquiring DR-TB is through the spread of drug resistance (DR) mycobacterial strains in the community. Individuals who have had contact with someone who has drug-resistant TB are more likely to contract the disease. As a result, assessing the major associated risk factors contributing to the emergence of DR-TB using data from the local setting is critical in order to take appropriate action. Objective: The study aimed to investigate the prevalence of drug-resistant tuberculosis and associated risk factors among TB patients from health care facilities in the Northern Region of South Africa. Methodology: In order to determine the prevalence of drug-resistant TB and associated risk factors, this investigation adopted a cross-sectional study design. The study enrolled 50 tuberculosis patients of age 18 years and older from healthcare facilities in the Vhembe region between August 2022 and August 2023. Patients undergoing active TB treatment were recruited as participants in this study. Data on demographic characteristics, comorbidities, healthcare-seeking behaviour, TB-related stigma, and adherence to infection control measures were collected using a detailed questionnaire. Allplex™MTB/MDR/XDR assay (Multiplex PCR) was used for rapid detection of M. tuberculosis and drug-resistant TB. To study the association between risk factors and development of drug-resistant TB, Rstudio was employed (a statistical analysis software package). The chi-squared method was used to determine whether the association between two qualitative variables were statistically significant among the study population. Results: Among the 50 sputum samples that were collected for the study, 17 (34%) remained TB active. Of these, 13 (26%) did not develop any resistance and the remaining 4(8%) presented drug-resistant TB strains which were resistant to at least one or more anti-TB drugs. There was an equal distribution in terms of gender. Four percent (4%) of the female cases had mono-resistant TB, of which 1(2%) was isoniazid resistance (INH) and the other one being 1(2%) rifampicin resistance (RIF). Furthermore, 4% of the male cases had Extensively drug-resistant TB (XDR-TB), showing resistance to rifampicin (RIF) + isoniazid (INH) + fluoroquinolone (FQ). Having HIV infection (P=0.042), unemployment (P=0.023), non-adherence to TB treatment (P=0.042), smoking habit (P=0.023), history of contact with DR-TB patient (P=0.023) and history of previous anti TB treatment (P=0.023) were found to be significant factors for the development of drug resistance. Other factors such as age (P=0.127), gender (P=0.127) and education (p=3.246) were not significantly associated with drug resistance TB. Conclusion: This study showed a low prevalence of drug-resistant TB in the Vhembe district with factors such as history of TB treatment, close contact with drug-resistant TB patients, non-adherence to TB medication, HIV infection and tobacco smoking, showing a significant association with drug-resistant TB. Hence, the study recommends that these factors must be taken into account in the planning and development of drug-resistant TB policies in order to work successfully towards the achievement of sustainable development goal of reducing TB by 80% before 2030. This study also highlighted that drug-resistant TB was more prevalent among the young and economically active age groups (20-40 years) than older individuals. Even though the prevalence of drug-resistant TB in this study was considered low, these findings highlight the need for health education efforts to strengthen accurate information to improve TB knowledge and correct misconceptions about TB among patients within the community especially the young age group.Item Open Access A metagenomic snapshot of selected wastewater treatment plants in Vhembe Region, Limpopo, South Africa: Investigating the resistome(2024-09-06) Jacobs, Damien; Potgieter, N.; Traore, A. N.Background: Water is crucial for human life. Rural communities often rely on natural water sources which may become polluted by wastewater due to various activities such as domestic use and agriculture. Antibiotic resistance genes (ARGs) may be transferred from wastewater to the environment and pose a global challenge they affect both human and animal-related sectors. Studying antibiotic resistance in wastewater treatment plants within Vhembe offers a representation of antibiotic resistance genes from entire communities. Knowledge of antibiotic resistance circulating in Vhembe has been sparsely studied. Metagenomics approaches allow for a broad overview of the resistome and the bacterial communities within environmental samples. Aim: To perform wastewater surveillance of antibiotic resistance genes and associated bacteria within Vhembe, Limpopo, South Africa, using a metagenomics approach. Method: A total of 32 sample duplicates were collected from the influents (n=18) and the effluents (n = 14) from nine wastewater treatment plants (WWTPs) around the Vhembe region, Limpopo, South Africa. One hundred milliliter was filtered using sterile cotton gauze and Wattman filter paper to remove debris and membrane filtered through 0.22um membrane filters to capture the bacteria within each sample. DNA was extracted directly from the 0.22 μm filters using a DNA miniprep kit. DNA was quantified using a spectrophotometer. Shotgun 18 metagenomic sequencing was performed on DNA extracts. Open-source bioinformatics pipelines were used to process and analyze raw sequence data, uncovering information of the bacterial community composition and associated ARGs in wastewater. Results: Site observations reveal animal and human activities within and near the sites. ARG analysis revealed an overall number 0f 220 ARGs detected across the WWTPs. Thirty-six genes were common to influent samples and 16 in within effluent samples, encoding predominantly against macrolides, sulfonamides and tetracyclines, beta-lactamases, and aminoglycosides. Some unique ARGS were detected at sites near South African borders. Bacterial Diversity showed the predominance of some genera, such as Arcobacter, Aeromonas, Pseudomonas and Acinetobacter. Pathogens were predominantly enteric and pulmonary, with some being linked to animals in past studies. A notable increase in some members of Mycobactericeae, among other bacteria, was noted in effluents.