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Item Embargo 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 Embargo 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 Embargo 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.Item Embargo Genetic divesity of Human Herpesvirus Type 8 in Northern South Africa(2024-09-06) Raphalalani, Mulalo; Bessong, Pascal Obong; Mavhandu-Ramarumo, Lufuno GraceBackground: 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 Embargo Microbial, metabolic and molecular determinants of neonatal mortality in the rural areas of Limpopo Province, South Africa: umblical cord blood metabolomic profile and bacterial anaysis and their association with perinatal complications(2024-09-06) Moagi, Innocent; Samie, A.; Maputle, M. S.; Mabasa, L.BACKGROUND: Neonatal mortality continues to pose a substantial public health challenge globally, particularly in developing countries. Some primary contributors are microbial infections, adverse pregnancy and birth outcomes (APBOs), and labor or obstetric complications. Despite this recognition, the underlying molecular mechanisms of these infections and perinatal complications are poorly understood. Interestingly, microbial and metabolomic approaches have emerged as effective techniques for the early detection and identification of biomarkers for microbial infections and perinatal complications, shedding more light on understanding the underlying molecular mechanisms of these adverse outcomes. Hence, this study employed microbial analysis (through 16S rRNA PCR assay and antibiotic susceptibility test profiling) and metabolomic profiling of umbilical cord blood to identify potential biomarkers for perinatal complications. METHODS: A cross-sectional study utilizing purposive sampling was conducted at selected district hospitals in the Vhembe district from May 2023 to September 2023. Following participants' consent, questionnaires were used for data collection and umbilical cord blood samples were collected from 129 participants. Blood culture was done, and antibiotic susceptibility testing was conducted on the isolates. Additionally, genomic DNA was isolated from the blood samples for the detection of pathogenic bacterial strains, using a conventional PCR assay. Untargeted metabolomic profiling approach using Liquid Chromatography-Quadrupole-Time-of-flight Mass Spectrometry (LC-q-tof-MS) was conducted. The data were entered into REDCap and exported to IBM Statistical Package for the Social Sciences (SPSS) software, version 26, for analysis. Descriptive analysis and multivariate logistic regression analysis were conducted, with statistical significance set at a p-value less than 0.05 at a 95% confidence level. For metabolomic analysis, multivariate analysis approaches were employed to identify signals that differed between perinatal complications and uncomplicated pregnancies. Metabolic pathway analysis of perturbed metabolites was conducted using MetaboAnalyst 6.0. RESULTS: Among 129 participants, the overall occurrence of perinatal complications stood at 35/129 (27.13%). The majority of participants, accounting for 55/129 (42.64%), fell within the age range of 20 to 29 years, while a significant portion (83.7%), were unemployed. Factors such as maternal age, birth weight, maternal blood pressure, anesthesia use, and delivery mode showed associations with perinatal complication risk, with corresponding P-values of 0.032, 0.027, 0.041, <0.001, and <0.001, respectively. Additionally, bloodstream infection (bacteremia) prevalence, as detected by culture-dependent and 16S rRNA PCR assays, was recorded at 30.23% and 26.36%, respectively. The majority of culture-confirmed bacterial isolates (58.06%) were gram-positive, with S. epidermidis (36.73%) and S. aureus (20.43%) being the predominant strains. Notably, E. coli infections showed a significant association with perinatal complications (P = 0.001). Bloodstream infection (Bacteremia) correlated significantly with maternal educational level, maternal blood pressure at birth, gestational booking stage, and pre-pregnancy BMI. Most culture-confirmed isolates exhibited high levels of antibiotic resistance to ampicillin, ceftazidime, and cefoxitin, while gentamicin, imipenem, amikacin, and ciprofloxacin proved effective against both gram-positive and gram-negative isolates. Furthermore, 93.38% of the tested isolates displayed multidrug resistance (MDR). Through untargeted metabolomic profiling analysis and multivariate analysis using the orthogonal partial least squares discriminant analysis (OPLS-DA) model, 107 metabolites were identified, showcasing differences between perinatal complications and uncomplicated pregnancies. Among the 107 perturbed metabolites, univariate analysis highlighted 50 upregulated and 57 downregulated metabolites in perinatal complications at P<0.05. Furthermore, the affected metabolic pathways, including ethyl lipid metabolism, sphingolipid metabolism, and glycerophospholipid metabolism, were identified as statistically significant. CONCLUSION: Our study revealed a high occurrence rate of perinatal complications (25.58%), with maternal high blood pressure, maternal age (10-19 years), low birth weight, delivery mode, and anesthesia for C-sections identified as significant contributors. Proactive healthcare interventions during antenatal care visits are crucial to minimize complications. Additionally, when looking at microbial analysis, there was no significant association between bacteremia and perinatal complications but highlighted a higher prevalence of bloodstream infections, linked to factors like maternal education level and BMI. Furthermore, upon conducting metabolic profiling, it was evident that specific umbilical cord blood processes were closely associated with perinatal complications, indicating their potential as biomarkers for assessment, prediction, and early intervention strategies.Item Embargo Synthesis and Characterisation of Metallic Nanoparticles from Medicinal Plant Extracts and their Application in combating Antibiotic-Resistant Bacterial Pathogens(2024-09-06) Nemaguvhuni, Murunwa Felicia; Samie, A.; Traore, A. N.; Razwinani, M.Background: The rise of antibiotic-resistant (ABR) bacterial pathogens is a major global health concern, especially in the case of respiratory tract infections (RTIs), which can make treatment more difficult. According to statistics, more than 2 million lives were lost due to ABR bacterial infections in 2019, highlighting the seriousness of the situation. This study aims to propose a novel approach to combating ABR bacteria by synthesising metallic nanoparticles (MNPs) from medicinal plants. Integrating nanoparticle-based strategies with medicinal plants demonstrates considerable promise in addressing RTIs. Methodology: The present study investigated the extracts of Spirostachys africana (S. africana), a medicinal plant traditionally used by healers to treat various infections. Extracts were prepared using methanol, ethanol, acetone, and distilled water. A comprehensive approach was employed to synthesise and characterise silver (SA-AgNPs) and gold (SAAuNPs) nanoparticles from S. africana extracts. Methods of characterisation included Ultraviolet-Visible spectrophotometry (UV-Vis), X-ray diffraction (XRD), Dynamic light scattering (DLS), Transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), and Liquid chromatography and mass spectroscopy (LC-MS). The antimicrobial activity of the nanoparticles and extracts was tested against the World Health Organization (WHO) ABR priority pathogens using agar well diffusion and microdilution assays. We further, assessed the cytotoxicity, anti-inflammatory, and antioxidant activities using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Griess assays, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, respectively. Results: The UV-Vis analysis revealed surface plasmon resonance (SPR) peaks at 480 nm and 541 nm, indicating the formation of SA-AgNPs and SA-AuNPs, respectively. XRD showed face-centred cubic structures with crystalline sizes ranging from 9–19 nm for SA-AgNPs and 9–10 nm for SA-AuNPs. The DLS measurements displayed a polydisperse distribution for SAAgNPs, while a monodisperse distribution was observed for SA-AuNPs. Most NPs aggregated over time, except for bark-methanol conjugated SA-AgNPs, which exhibited more stability with a zeta potential value of -27 mV. The TEM images showed particle core sizes of 5–49.5 nm for SA-AgNPs, predominantly spherical, and 6–32 nm for SA-AuNPs, mainly spheroidal. The FTIR analysis identified functional groups including hydroxyl, carboxyl, and amine groups in both plant extracts and SA-AgNPs/SA-AuNPs. These fuctional groups are involved in the reduction and capping of Ag+ and Au+ ions to form SA-AgNPs and SA-AuNPs, respectively. The LCMS technique identified 23 bioactive compounds from S. africana extracts, with flavonoids being the most dominant. Antimicrobial assays using agar well diffusion and microdilution methods showed SA-AgNPs were more effective than crude extracts and SA-AuNPs against tested ABR bacteria. Acetone-conjugated SA-AgNPs having the highest zone of inhibition (22 mm) against P. aeruginosa. In contrast, methanol/ethanolconjugated SA-AgNPs displayed potent antimicrobial activity (MIC = 0.05 mg/mL) against E. coli, P. aeruginosa, and A. baumannii, respectively. Additionally, Acetone extracts exhibited selective toxicity against MCF-7 cancer cells, while stimulating MCF-10 cells and RAW 264.7 macrophage cells at concentrations of 0.078 mg/mL. Furthermore, ethanol extracts and ethanol-conjugated SA-AgNPs/SA-AuNPs significantly decreased NO production, indicating their potential as anti-inflammatory agents. Acetone extract exhibited excellent antioxidant activity (IC50 = 0.000335 mg/mL). Conclusions: The MNPs synthesised from S. africana extracts offer a promising solution to combat ABR bacterial pathogens, particularly those causing RTIs. The S. africana extracts and MNPs show significant cytotoxic, anti-inflammatory, and antioxidant activities.Item Embargo Norovirus-host interaction studies HBGA phenotypic and genotypic profiles of paediatric patients with diarrhoea from rural communities of Limpopo South Africa(2024-09-06) Khumela, Ronewa; Potgieter, N.; Traore, A. N.; Kabue, J. P.Human norovirus (HNoV) are the leading aetiological agents of viral acute gastroenteritis (VAGE) worldwide. Approximately 685 million cases of VAGE and 219 000 deaths associated with norovirus infection are recorded each year. The burden is more severe in developing nations such as those in Africa, and children below the age of five are the most vulnerable. Noroviruses are commonly transmitted from person-to-person via the faecal oral route and contaminated fomite, food and water. Norovirus are extremely prevalent and exhibit great genetic diversity and rapid rates of evolution. Studies have shown that histo-blood group antigens are essential genetic susceptibility factors for HNoV infection. Histo-blood group antigens (HBGAs) are a class of carbohydrates antigens found in the surface of red blood cells and bodily fluids or tissues. HNoVs recognize and bind to HBGAs for attachment to human epithelial cells in the gastrointestinal tract. The synthesis of HBGAs is mediated by fucosyltransferase and glycosyltransferase, which are genetically regulated by FUT2 (Secretor), FUT3 (Lewis) and ABO (H) genes. Individuals with functional FUT2 gene are termed secretors; they express HBGAs in gut epithelial cell surface and body fluids. Natural variability and inability to express HBGAs (due to mutations) plays a role in population genetic susceptibility to norovirus infection and genotype distribution. Approximately 20% of VAGE are caused by norovirus. Recently, global prevalence of norovirus was reported at 16%. In African countries, the range of reported prevalence was between 13% and 20%. However, higher prevalence has been reported, particularly in Ghana and South Africa. Previous data in South Africa have demonstrated the occurrence of norovirus, with higher number of cases reported in rural communities. There are limited or no studies to explain the elevated prevalence of enteric HNoVs in rural communities of Limpopo, South Africa. Various factors including genetic, geographical, meteorological and socio-economic may play a role in the epidemiology of enteric viruses. A potential interaction between human and viral genetic diversity has been demonstrated via association of population secretor profiles and viral genotype susceptibility and distribution. Therefore, this study, aimed to investigate the prevalence and norovirus host genetic susceptibility profiles in paediatric population under five years, within rural communities of South Africa. To achieve the aim of this study, the following objectives were set: 1) To determine molecular characteristics of HNoV strains circulating in the rural communities of Vhembe district, South Africa; 2) To determine host phenotypic profiles of HBGAs (ABO/H and Lewis system) in HNoV infections among children from rural communities of Vhembe district, South Africa; 3) To determine the host genotypic profiles of HBGAs (FUT2/FUT3) in HNoV infections among children from rural communities of Vhembe district, Limpopo Province (South Africa) and 4) To assess the correlation between circulating HNoV genotypes and HBGAs profiles in young children from rural communities of Vhembe district, Limpopo Province (South Africa). To achieve the study objectives, a cross-sectional survey in children with (200) and without (100) diarrhoea, below the age of five was performed. Co-paired stool (300) and saliva (300) samples were collected from October 2019 to September 2021, in South African healthcare institutions (clinics and hospitals) within the Vhembe district of Limpopo province. Objective 1: HNoVs were detected from stool samples using real-time RT-PCR, then amplified by conventional RT-PCR, and further genotyped by Sanger sequencing. Norovirus strains from this investigation were compared to the worldwide circulating strains by phylogenetic analysis using MEGA11. ClustalW software was used to compare nucleotide similarity among strains genotyped in this study and those previously obtained within the same study region. Objective 2: HBGAs phenotypic profiles were determined from saliva samples by ELISA assays. Monoclonal antibodies including A, B, Le-a, and Le-b were used to determine HBGAs phenotypic profiles in saliva samples. In samples where the phenotypic status was inconclusive, the secretor and non-secretor phenotypes were further confirmed using the Ulex europaeus agglutinin (UEA-1), which is specific for Fucα1-2Gal-R present in secretor, but not in non-secretor saliva. Objective 3: Genotypic characterization of HBGAs were determined by Touch-Down-PCR using the Platinum Taq DNA Polymerase High Fidelity enzyme. The primers used to detect SNPs were also used for nucleotide sequencing of both FUT2 and FUT3 partial genes of selected samples (100). Objective 4: Statistical analysis was performed using Graphpad 10.0 for the association of HNoV prevalence and HBGAs profiles. The results indicate that HNoVs were still highly prevalent in children from Vhembe district, with a confirmed significant difference (p < 0.0001) between symptomatic (37%) and asymptomatic (14%). Genogroup II noroviruses predominated children with AGE (80%), whereas genogroup I norovirus were high in children without AGE. Genotype GII.4 Sydney 2012 [P31] were dominant throughout the study period. The detected strains were phylogenetically closely related to the worldwide circulating norovirus strains but showed low nucleotide similarity when compared to other HNoV strains previously characterized in Vhembe region. Elisa assay findings showed diversity of HBGA phenotypic profiles including Lewis and ABO type amongst study participants. Both Lewis negative and positive phenotypes were identified with the latter being the predominant and mostly infected with HNoV. All ABO phenotypes were also identified within the study population. The highest occurring was type O, which also represented the primarily norovirus infected group, followed by type A. Overall, majority of children had secretor phenotypes (81%) over non-secretors (19%), similarly, higher cases on HNoV were detected in children with secretor phenotype (93%). Amongst the selected samples for FUT2 genotyping, only 42 could be amplified and of those, 36 (36%) were successfully genotyped. Majority of FUT2 secretors were homozygous mutations (64%) compared to heterozygous (28%). However, only 8% were determined as non-secretor with homozygous nonsense mutation on the G428A position. FUT3 genes were also associated with norovirus positivity. The G508A SNP was the most common and predominant among positive norovirus cases. Interestingly, 2 mutant SNPs were detected. Although non-secretor was the lowest in HBGAs genotypic profiles and is often related to resistance to infection, norovirus GII.4 Sydney 2012 [P31] were identified in two samples from hospitalized children. Overall, the data revealed correlation between the presence of HNoV and the population HBGAs profiles. Individuals expressing HBGAs of secretor status were highly infected with norovirus, whereas non-secretor individuals demonstrated protection or less susceptibility towards norovirus infection. The GII.4 Sydney 2012 [P31] recombinant strain was dominant, followed by the GII.4 Sydney 2012 variant in all the secretor profiles. This study concludes that the prevalence and susceptibility of human norovirus infection in symptomatic and asymptomatic children from rural communities of Vhembe District is related to their HBGAs genetic profiles. The dominance of GII.4 Sydney 2012 [P31] in AGE children may be explained by the strain’s epidemiological fitness, and genetic susceptibility related to the prevalence of secretor profiles. Population’s genetic profiles may potentially drive human norovirus genetic diversity and evolution. This has implications for vaccine formulation in the current era of vaccine development. Further surveillance on impoverished communities where preventive strategies will most likely have greater impact is needed to monitor distribution of the virus and overall population HBGAs status.Item Open Access Determination of the impact of Antiretroviral therapy in the proportion and genetic diversity of diarrheal associated gut microbiota among HIV infected population(2023-10-05) Musetsho, Phumudzo Pertunia; Mavhandu - Ramarumo, L. G.; Bessong, P.O.Background: Human gut microbiota are microorganisms that reside in the gastrointestinal tract of humans. Gut microbiota provide various functions in the gut including protection from invasion of pathogenic microbes, shaping host immunity and substrate metabolism. HIV which targets the CD4+ T cells in the gut associated lymphoid tissue (GALT), causes gut microbiota alteration due to disruption of the gut barrier. This result in microbial translocation and pathogen invasion which lead to decreased diversity and proportion of gut microbial and increase in pathobionts and pathogens. As a result, HIV infected patients suffer from diarrhoea due to compromised gut microbiota. Antiretroviral therapy (ART) suppresses viral replication and lead to an undetectable viral load, thus restoring the immune system. Previous studies show that ART does not entirely restore the depleted gut microbiota, hence this study aims at determining the change in proportion and genetic diversity of diarrheal associated gut microbiota in HIV infected patients. Hypothesis: Antiretroviral therapy causes changes in the proportion and diversity of diarrheal causing gut microbiota Objective: The objective of the study was to determine the impact of ART in the proportion and genetic diversity of gut microbiota among HIV infected patients. Methodology: Paired blood and stool samples were collected from 17 HIV infected and 11 HIV uninfected individuals (controls). Samples were collected during baseline (before ART initiation); and samples were collected after every three months thereafter, for a total of 12 months. Blood samples were used for CD4 and viral load measurements using BD FACSPresto machine (BD Biosciences) and HIV Qualitative PCR, respectively. Stool samples were used for extraction of total nucleic acid (TNA) using a modified Qiagen QIAamp Fast DNA Stool Mini Kit. Purification of TNA was done using Ampure XP bead. DNA library preparation kit (Illumina) was used for DNA library preparation. Illumina miniseq sequencing was used for sequencing and the obtained sequence reads were analysed using Geneious prime software for trimming and filtering low sequence reads and to determine the genetic diversity of diarrheal causing gut microorganisms. DRAGEN metagenomics was used for taxonomical classification to generate the proportion of diarrheal causing gut microorganism. GraphPad prism was used to generate graphs and for statistical analysis to generate the association between CD4+ T Cell/viral load and diarrheal causing gut microbiota. Results: Escherichia coli (82.33%), Bacteroides fragilis(2.46%), Shigella spp.(1.96%), Salmonella enterica (1.07%), Clostridioides difficile(0.99%), Campylobacter jejuni (0.20%) were the most prevalent detected microoganisms from HIV infected individuals at prior ART initiation. Among HIV negative individuals, Escherichia coli (62.52%), Bacteroides fragilis(18.43%), Shigella spp.(0.40%), Salmonella enterica (3.05%), Clostridioides difficile(15.06%), Campylobacter jejuni (0.54%) were found. The proportion of Escherichia coli (90.77%), Clostridioides difficile (2.20%), Shigella spp. (2.73%),and Salmonella enterica (1.28%) had increased with treatment at three months post treatment in HIV infected individuals. At six months post ART, there was an increase in the proportion of Bacteroides fragilis (88.65%) and Vibrio cholera (0.01%) when compared to HIV positive samples at three months post-treatment. Escherichia coli (9.74%), Cryptosporidium parvum (1.74%), and shigella spp. (0.35%) increased with treatment from six months to nine months post-treatment. Escherichia coli (58.58%), Shigella spp. (0.81%) and Clostridioides difficile (24.77%) had increased with ART at twelve months post treatment. The proportion of diarrheal causing gut microbiota were fluctuating throughout the intake of ART at different time points. There was no significant correlation between CD4/viral load and diarrheal causing microorganisms, as a result ART did not have an effect in the proportion of diarrheal causing gut microorganisms. Genetic diversity of diarrheal causing gut microbiota was higher in HIV positive individuals prior ART than in HIV negative individuals except for Salmonella typhi. Among HIV infected individuals, genetic diversity of most diarrheal causing gut microbiota was higher at three months post treatment. The fluctuation of diarrheal causing gut microbiota’s genetic diversity from six months to twelve months post treatment may be due to inconsistent change in number of viral loads and CD4 counts at different time points of ART. However, this was statistically insignificant. As a result, the change in the genetic diversity of diarrheal causing gut microorganisms was not due to ART. Conclusion: ART does not eradicate diarrheal causing gut microbiota. However, ART encourages a low genetic diversity of diarrheal causing gut microbiota.Item Open Access Investigating the therapeutic effects of silver and gold nano particles capped with selected medicinal plants against tuberculosis(2023-10-05) Motene, Matshoene Violet; Traore, A. N.; Potgieter, N.; Mavumengwane, V.Background: Tuberculosis (TB) is implausibly still considered as one of the leading causes of death, in the 21st century. Despite the curative treatments and measures of control in the communities, TB plays a significant role in human infectious disease. Studies have shown that there is an emergence of major drug-resistant TB. Aim: This study was aimed at evaluating the biological activities of silver and gold nanoparticles capped with selected medicinal plant extracts against Mycobacterium tuberculosis. Methodology: P. africanum and Z. mucronata barks and leaves were collected in the Vhembe district of Limpopo. Plant phytochemical constituents were extracted using distilled water and absolute methanol. The silver nanoparticles (AgNP) and gold nanoparticles (AuNP) were capped using crude extracts through the process of green synthesis; and were characterized by Ultraviolet-Visible spectrophotometry (UV-VIS), and transmission electron microscopy (TEM). Metabolites present in the plant extracts were profiled using liquid chromatography mass spectrometry (LC-MS). Cytotoxicity activity of plant extracts and nanoparticles were determined by MTS assay on HEK293 cells. The anti-inflammatory activity was determined through the nitric acid synthase (NOS) inhibitory test. The antimycobacterial activity was determined using microbroth dilution. Results: Following extraction by maceration, methanol was able to yield more extracts compared to distilled water due to their differences in polarity. The selected plants were found to contain numerous antioxidant significant for anti-inflammatory and cytotoxic activity. The metabolites play a role in the formation of the nanoparticles. Plant extracts and gold nanoparticles had little impact on the cell lines, thus were concluded to be non-toxic. Whilst silver nanoparticles exhibited toxic activity on the cell line at both concentrations, hence were considered toxic to human. Silver nanoparticles of ZML and ZHL, and gold nanoparticles and plant extracts of PML exhibited anti-inflammatory activity at 100 μg/mL, whereas PML was able to decrease nitrite concentration at both concentrations. Conclusion: Adoption of TB strategies recommended by World Health Organization (WHO) to reduce TB deaths and incidence rate by 90% and 80%, respectively, (less than 20 TB cases per 100 000 population).Item Open Access Development of Liquid Chromatography Mass Spectrometry technique for evaluation and differentiation of plant derived isomers(2023-10-05) Mbedzi, Dakalo Terrence; Madala, N. E.; Mathomu,L. M.; Mhlongo, M. I.Cinnamic acid containing molecules have been shown to possess unprecedented pharmacological activities against various physiological disorders. Most of the plants containing these compounds have been shown to exhibit multiple bioactivities, notably against various diseases which are currently without a cure, for example human immunodeficiency virus (HIV) associated ailments. As in other plants, most of the pharmacologically relevant metabolites are produced in minute concentrations, and, as such, alternative means to produce high levels of these compounds are imperative. Herein, UV-induced photochemical conversion of cinnamic acid containing molecules is presented as a feasible means to diversify the number of bioactive metabolites in plant extracts. Naturally, UV rays from sunlight were shown to result in the formation of geometrical isomers. The presence of these isomers has resulted in analytical challenge, which has resulted in erroneous identification and isolation of an active isomer. Virtual screening and docking studies provide insights into the potential interactions between molecules and their targets, and can be useful in predicting the biological activity of compounds. This study further shown that synergistic isomerism could be beneficial during inflammation conditions, and it suggests that certain isomers of a compound may work together to enhance the desired therapeutic effect. Herein, liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) method capable of distinguishing between these isomers was developed.Item Open Access Assessment of knowledge, attitude and practices of small household farmers towards heartwater disease and molecular characterization of Ehrlichia ruminantium in Limpopo province, South Africa(2023-10-05) Mathebula, Doris; Samie, A.; Sigidi, M. T.Background: Heartwater is a disease spread by ticks that is brought on by the obligatory intracellular bacterium Ehrlichia ruminantium. Heartwater disease is one of the major obstacles to livestock production as it affects many livestock animals including domesticated animals such as goats, cattle, and sheep as well as wild ruminants. In Southern Africa, it is spread by Amblyomma hebraeum ticks, and in the rest of Sub-Saharan Africa, it is spread by A. variegatum ticks. In this study, epidemiological and molecular features of Ehrlichia ruminantium in Mopani and Vhembe Districts, from ticks isolated from cattle, goats and sheep were investigated. Method: A total of 121 small household farmers from different villages in the Vhembe and Mopani Districts were recruited in the study after they have signed an informed consent. The participants were interviewed using a questionnaire to collect data related to their knowledge, attitude and practices towards heartwater disease. Ticks were collected from cattle, goats, and sheep belonging to 48 households, yielding a total of 244 ticks. Genomic Deoxyribonucleic acid was extracted from the ticks and analysed using real-time qPCR assay targeting a 226bp fragment of the PCS20 gene. Finally, a number of samples were sent for sequencing to identify different strains circulating in the region. Neighbor-joining method was used to infer phylogenetic positions on the basis of 16S rRNA gene. Results: According to the findings of the questionnaire evaluation, only about 23.1% of the participants had some knowledge of heartwater disease. Furthermore, the highest proportion of the study population (76%) associated heartwater with air-borne transmission and 77.7% of the participants failed to identify the season in which heartwater commonly occurs. About 69% of the respondents associated ticks with animal diseases while 49.6 % correctly highlighted that, ticks are disease carriers. Farmers had a positive attitude towards control and treatment of heartwater by stating that they would use prescribed medicine 23.9%, vaccines 7.4% or consulting a specialist 2.5%. Few farmers indicated that they use homemade mixtures (0.8%), dipping and spraying (0.8%) to manage animal diseases. The most preferred method of tick control used by farmers was spraying of acaricide treatment 63.6%. On account of the poor animal services reported among the visited rural communities, some farmers opted for removing ticks by hand 28.9% as their supplementary tick control method. Majority of the farmers fed their livestock at the bush 68.6% which was the most contributing factor to increased tick infestation as reported by farmers. Several plants used as medicine to treat various animal diseases were mentioned. These included: Melia azedarach, Albizia adianthifolia, Gymnosporia senegalensis, Dicerocarryum senecioides etc. The type of disease affecting the livestock mentioned by the participants was diarrhoea (43.0%) which is among the list of heartwater symptoms. The study demonstrates that respondents had inadequate knowledge about heartwater disease. Animal services need to be upgraded in order to help the farmers improve the quality of their produce. The results of the PCR test showed that 56.2% of the household farms had E. ruminantium infection. The distribution of E. ruminantium by source of ticks and animal type revealed that cattle are more prone to tick distribution 43.5% as compared to other animal sources of ticks. Nzhelele municipality had the highest prevalence of E. ruminantium (37.0%) compared to the other municipalities. Prevalence of E. ruminantium by gender of farmers was found to be high from males 36.7% than females 25.0%. Farmers with household income > R20000 had the highest prevalence of E. ruminantium (50.0%) than farmers with household income < R500, (14.3%). The age of the farmers 15 – 20 years old revealed the highest prevalence 46.7 % of E. ruminantium infection. However, there were no infection among the ticks obtained from animals belonging to farmers above 60 years old. Farmers who had tertiary level of education showed the highest prevalence 46.8% probably because of limited time to attend to their livestock. Many clades were identified by phylogenetic analysis of the E. ruminantium PCS20 genotypes. Conclusion: This study showed that there is need to further educate small farmers on heartwater disease. It also showed that indigenous knowledge still contributes significantly to the management of animal diseases in most rural communities. The application of real time PCR showed a high prevalence of E. ruminantium infected A. hebraeum ticks from livestock and should be considered in the continuous monitoring of the animal population in order to avoid heartwater disease outbreak in the communities which could be detrimental for the local economy. Furthermore, future vaccine development against E. ruminantium should consider the diversity observed in the present study. That could be helpful in managing heartwater disease in the areas that were investigated.Item Open Access Cytotoxicity and anti-mycobacterial activities of sclerocarya birrea and Dodonae viscosa anguistifolia against MTB strain(2023-10-05) Marole, Tsireledzo; Traore, A. N.; Potgieter, N.; Mavumengwane, V.Background: Tuberculosis is a major public health concern with over 2 billion people currently infected, 8.6 million cases per year and more than 1.3 million deaths annually. Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB) which mainly affects the lungs causing pulmonary TB. In South Africa, the use of the Directly Observed Treatment Short Course (DOTS) strategy is applied where patients have to travel to primary Health care facilities (DOTS clinics) to get their medication. The rising prevalence of multidrug resistant and extensive drug resistant TB throughout the world highlights the critical need for novel anti-tuberculosis compounds/drugs. Objective: The aim of this study was to determine the cytotoxicity and antimycobacterial activities of Sand Olive and Marula. Methodology: The leaves (L) and barks (B) of Dodonaea Viscosa Anguistifolia (DVA or D) and S. birrea (S) plants were collected in the Vhembe district, Limpopo. Separate macerations in methanol (C) and distilled water (H) were done to obtain a total of 8 crude extracts (SBC, SBH, SLC. SLH, DBC, DBH, DLC, DLH). Phytochemical analysis was conducted on all extracts using thin layer chromatography (TLC), the profiling of metabolites was achieved with liquid chromatography-Mass spectrometry (LCMS) method. The antimycobacterial activity against M. smegmatis was determined using the microbroth dilution assay. The cytotoxicity of the plant extracts was assessed using the MTT assay. Results: The percentage yield ranged from 21.07% (DBC) to 4.4% (DBH). The phytochemical screening showed the presence of saponins, cardiac glycosides, steroids, terpenoids and tannins. The BEA solvent system revealed more bands than the CEF solvent system while the EMW solvent system was the least efficient. LCMS method showed sufficient revolving power to separate isomeric forms of several compounds. From the extracts of DVA and S. birrea, 508 chemicals were present in all the chromatograms assessed, but only 40 compounds were putatively identified and comprised of flavonoids, phenolics, terpenoids, and saponins. Of the 8 plant extracts that were tested 5 (DLH (50μg/ml (83.5%), (100μg/ml (85.9%)), DBH (50μg/ml (70.9%), (100μg/ml (60.3%)), DBC (50μg/ml (76.6%), 100μg/ml (83.9%) SLH (50μg/ml (66.8%), (100μg/ml (66.1%) and SLC (50μg/ml (79.9%), (100μg/ml (77.1%)) were found to have moderate cytotoxic effects on Vero cells at both treatment concentration while 3 (DLC (50μg/ml (91.5%), (100μg/ml (105%), SBH (50μg/ml (98.7%), (100μg/ml (106.8%) and SBC (50μg/ml (105.3%), (100μg/ml (118.2)) did increase viability of Vero cells. Amongst all the samples screened for antimycobacterial activity, only 3 plant extracts (SBC, DBC and DBH) inhibited the growth of M. smegmatis. Conclusion: The results of the study demonstrate the potential use of DVA and S. birrea as alternative treatment for tuberculosis. The bark of the plants may contain active compounds with anti-TB activities needing further investigation.Item Open Access Microbial analysis of street vended ready-to-eat meat around Thohoyandou Area, Vhembe District, Limpopo Province, RSA(2023-10-05) Raedani, Tshimangadzo Jeanette; Musie, E. M.; Afsatou, TraoreBackground: Street-vended meats are meats that are prepared and sold by vendors on the street. Different types of street vended meats are chicken, pork, beef etc. Street vendors are an integral part of urban economies around the world, offering easy access to a wide range of goods and services in public spaces. Despite these benefits, meat has been well-known as a potential channel for spreading food-borne diseases due to its high-water activity, high protein content, and approximately neutral pH, which create favourable conditions for the multiplication and survival of pathogenic bacteria. Street foods are frequently associated with food-borne illnesses due to their exposure to contamination hence this reduces the quality of these meat. Meat sold by the street vendors could be the source of infectious pathogens and previous studies showed that there is high number of pathogenic bacteria found on meat. The aim of the study was to determine the microbial quality and safety of street vended ready-to-eat meat sold around Thohoyandou Area. Method: A total of 168 samples of street-vended meats consisting of chicken (n=84) and beef (n=84), were collected from the local street vendors around Thohoyandou area. Samples were selected using simple random sampling and purchased meat samples were transferred from vendor packaging into sterile lunch boxes at the point of purchase. The packed samples were placed in a cooler box and immediately transported to the Department of Food Microbiology laboratory, University of Venda for further analysis. Ten grams (10g) of chicken or beef samples were then transferred into a zip lock bags containing 90 ml of peptone buffered water and then cultured in different plate’s containing the selective media: MSA (Oxoid Ltd) was used to culture Staphylococcus aureus, Eosin Methylene Blue (EMB) for E. coli 0157, xylose lysine deoxycholate (XLD) agar, (Oxoid Ltd) for Salmonella, and Sorbitol McConkey (Oxoid Ltd) for Shigella, and then plates were incubated for 24 hours at 37oC. The presumptive colonies were then sub-cultured on Nutrient agar for purification and the plates were incubated at 37oC for 24 hours. The microorganisms were identified using Gram staining and biochemical tests (Catalase, API 20E and Klingler iron Agar Test, and Vitek 2 system). The antibiotic susceptibility was done to determine susceptibility of the microorganisms using antimicrobial Agent such as Ampicillin, Chloramphenicol, Penicillin, Neomycin, Tetracycline, Streptomycin and Amoxicillin). The molecular 5 characterization was done to determine different pathotypes of E. coli using multiplex PCR. Results: Out of 168 samples tested, 32 (19%) were found to be positive for Staphylococcus spp with highest percentage found in cooked chicken meat. The most prevalent staphylococcus species identified in this study were S. xylosus (13.2%) and S. saprophyticus (10.5%). The prevalence of E. coli was found to be 29 (19.3 %) in which highest percentage was found in fried chicken. The antibiotic susceptibility profile of E. coli isolated showed that 100% were Resistant to Ampicillin (AMP), Tetracycline (T) and penicillin (PG) and 100% were susceptible to Neomycin (N). Staphylococcus spp. isolates showed 100% resistance to Ampicillin (AMP) and 100% susceptible to Neomycin(N). The virulence genes ranged from 13,33% to 86,67% with asta, stx1, and eae being the most prevalent. The pathotypes that were detected in this study were EPEC, EHEC, ETEC, EAEC, and EIEC and majority of the isolates were positive for mixed pathotypes (contamination). Conclusion: This current study demonstrated that the microbial quality and safety of street vended meat is inadequate and therefore not acceptable for safe consumption. Therefore, it is essential to monitor the presence of microbes in meat and the detection of these organisms in all beef and chicken meats investigated serves as a warning of foodborne diseases that could be associated with poor personal hygiene, and poor food preparation.Item Open Access Assessment of microbial quality and safety of ground beef/product sold in differrent retailers around Thohoyandou Area, Vhembe District, Limpopo(2023-05-19) Mukosi, Fulufhelo Valentine; Musie, E. M.; Traore, A. N.Title: Assessment of microbial quality and safety of ground beef/product obtained from selected retailers around Thohoyandou area; Vhembe district; Limpopo. Background: It has been proven that animal products are easily contaminated with microorganisms, and this supports microbial growth if not properly handled, processed, and preserved. Ground beef and its wholesale products are becoming popular because of the demand for rapid meal preparation and services, especially in the fast-food industry. Despite the control measures in place, foodborne infections continue to be an immense problem, with millions of cases occurring annually worldwide. In South Africa, illnesses and deaths related to food consumption continue to be reported. In addition to the misery caused, the financial loss associated with meat spoilage and illnesses is enormous. Therefore, this study aimed to assess ground beef's microbial quality and safety in different retailers around Thohoyandou area, Vhembe District. Methodology: A total of 160 ground beef/product samples was randomly purchased from various retailers in Thohoyandou and transported on ice to the University of Venda microbiology laboratory for analysis. The potential microbes were cultured in enrichment media (peptone buffered water) for 5 minutes in room temperature. The culture was then sub-cultured in different plates containing selective media (e.g., EMB for E. coli, SS for salmonella and Shigella, MSA for Staphylococcus spp.) using the spread plate technique. Isolates were then identified by the Gram staining technique and biochemical tests such as Catalase, Urease, Citrate, Kligler Iron Agar, and VITEK system. Moreover, the antibiogram activity of isolated pathogens was screened against medically used and commercially available antibiotics. Furthermore, the DNA of the isolates was extracted, and multiplex PCR was conducted to determine different virulence genes and pathotypes. Hemolysin test was done in blood agar plates to identify virulence characteristics of E. coli isolates. Results: Out of 160 samples analyzed, E. coli was detected in 80 (50%), Staphylococcus spp. in 117 (73.12%), Salmonella in 60 (37.5%), and Shigella species in 108 (67.5%). Most Enterobacteriaceae (E. coli, Salmonella and Shigella) isolates were resistant to Ampicillin and Cefoxitin. Staphylococcus isolates showed high resistance to Cefoxitin (93.33%) and Oxacillin (93.33%). Out of 30 E. coli isolates subjected to mPCR assay, 23 isolates were of different pathotypes with EPEC (53.33%) being the most prevalent pathotype. Asta with 73.33% was the dominant virulence gene obtained. Thirty (30) E. coli isolates were tested for hemolysin activity and Alpha hemolytic activity was observed in 76.6% isolates, while beta hemolytic activity observed in 10% isolates. Some of the isolates presented non-hemolytic strains (13.3%). Conclusion: It was concluded that ground beef/products from established retailers were contaminated with pathogenic bacteria, and microbial quality was thus inadequate.Item Open Access Characterization of cervicovaginal HPV virome and bacteriome in HIV -i infected women in Northern South Africa(2023-05-19) Ratshilindela, Mpho; Bessong, Pascal Obong; Tebit, DenisBACKGROUND: The presence of a highly diverse bacterial species in the cervicovaginal niche is linked to a higher risk of contracting human immunodeficiency virus (HIV), persistent infection with human papillomavirus (HPV) and consequently cervical cancer. It is unknown how cervicovaginal bacterial species associate and interact with other viruses. This study hypothesized that HIV/HPV co-infected women have an increased diversity of cervicovaginal virome and bacteriome. OBJECTIVES: The study’s primary objective was to characterize cervicovaginal HPV virome and bacteriome in HIV-infected women from selected health care facilities in Northern South Africa. Specific objectives were to determine the presence of HPV virome in HIV-infected women and HIV-noninfected women, to determine the presence of bacteriome in HIV-infected women and HIV-noninfected women, and to determine the relational occurrence of virome and bacteriome according to HIV status. METHODOLOGY: Cervical swabs from 50 HIV/HPV co-infected women; 50 HIV-infected, HPV-noninfected women; and from 50 HPV-infected, HIV-noninfected women were used to extract total deoxyribonucleic acid (DNA). To determine HPV virome, total DNA was enriched by rolling circle amplification (RCA) using an illustra TempliPhi amplification kit. To determine bacteriome, a two-round polymerase chain reaction (PCR) was employed targeting a bacterial 16S rRNA gene. Amplification products obtained from RCA and PCR were purified and sequenced by Next Generation Sequencing (NGS) using a MiniSeq platform (Illumina). The quality of the sequences was validated using the FastQC program. Sequences of good quality were assigned to viral family and genera using the Dragen metagenomics online tool with BaseSpace Sequence Hub. Bacterial sequences were assigned and categorised into vaginal community state types (CSTs) using the Dragen metagenomics online tool with BaseSpace Sequence Hub. The relational occurrence of virome and bacteriome according to HIV status was assessed through Chi-square statistical analysis available in GraphPad Prism version 9.3.1. Differences in occurrence were expressed as probability (P)-values. RESULTS: A diverse group of viral families was observed among HIV/HPV co-infected women. Papillomaviridae (14/34; 41%) was the most prevalent (P<0.0001), followed by Herpesviridae and Phycodnaviridae (12/34; 35%) each, Poxviridae (10/34; 29%), Mimiviridae (7/34; 20%), Maiseilleviridae (3/34; 9%) and Anelloviridae (2/34; 6%) in order of decreasing prevalence. A highly diverse group of bacteriophages was observed, with Myoviridae (31/34, 91%) being the most prevalent (P<0.0001). Among HPV-infected HIV-noninfected women, v Papillomaviridae (8/26; 31%) was the most prevalent (P<0.0001), followed by Anelloviridae (4/26; 15%), Phycodnaviridae (4/26; 15%), Poxviridae and Herpesviridae (2/26; 8%) each in order of decreasing prevalence. Myoviridae (6/26; 23%) was the most prevalent bacteriophage family detected (P=0.0005). Among HIV-infected HPV-noninfected women, a small group of viral families was observed, with Myoviridae (3/11,27%) and Siphoviridae (3/11, 27%) being the most prevalent viral families detected (P=0.0005 each). HPV 16 was the most common high risk (hr) HPV type in HIV-infected women and HIV-noninfected women of this study. This genotype co-existed with other hrHPV or probable hr types including HPV 54, HPV 53, and HPV 26. Overall, hrHPV types were more prevalent in HIV-infected women than in HIV-noninfected women, although this difference was not statistically significant (P=0.2832). When the occurrence of hrHPV types were considered individually, HPV 54 occurred more significantly in HIV-noninfected women than in HIV-infected women (P<0.0003). Bacteriome revealed the presence of community state types (CSTs) one, two, three, four and five among study participants. Among HIV/HPV co-infected women, CST one (L. crispatus), CST three (L. iners) and CST four (high bacterial diversity) were observed, with CST four being the most prevalent. Among HPV-infected, HIV-noninfected women, CST one, three, four and five (L. jensenni) were observed, with CST four, also being the most prevalent. Among HIV-infected, HPV noninfected women, CST three and four were observed. CST three and four were associated with viral infections. Gardnerella vaginalis (75%), Pretovella spp (54%), Atopobium spp (50%), Bifidobacterium spp (27%), Porphyromonas spp (53%), Pseudomonas spp (50%), Faecalibacterium prausnitzii (47%) and Bacteroides spp (35%) were the most prevalent anaerobic bacterial species detected in CST four. A diverse group of bacterial families occurred more significantly among HIV-infected women as compared to HIV-noninfected women for NGS data of RCA products (P<0.0001) and NGS data of 16S amplification products (P<0.0001). CONCLUSION: In conclusion, this study showed a higher diversity of cervicovaginal virome and bacteriome in women who were HIV/HPV co-infected than in those singly infected with HIV or HPV. The relationship between viral infections and a high diversity of bacterial species (CST four) observed herein may be a useful indicator of the individual’s disease state, indicating the likelihood of developing cervical lesions and cervical cancer. As a result, additional research is necessary to uncover the association between viral infections and CST four with disease state. Vaccine development and antiviral research should also target compounds that boost the cervicovaginal environment and maintain vaginal homeostasis.Item Open Access Examining institutional (policy) and administrative framework for water and sanitation (WSS) services in Zimbabwean cities to inform development of a new framework(2022-11-10) Taonameso, Solomon; Potgieter, N.; Traore, A. N.; Mudau, S. L.The lack of access to water supply and sanitation (WSS) services in developing countries has caused various waterborne diseases and related millions of deaths. The challenges have compelled various countries to take note of the need to incorporate both technical and policy aspects of WSS services in order to promote the delivery of these services. This research examines the current institutional (policy) and administrative framework for water services in Zimbabwean cities in order to find insights that will inform the development of a new framework. The study examines the causes of urban water conundrums by identifying qualitative gaps between the ZNWP and its implementation using both an empirical and a secondary-based study; assesses the causes of urban water conundrums by focusing on quantitative gaps between the ZNWP and its implementation in the provision of water supply services using both an empirical and a secondary-based study; evaluates the current service level on water supply and sanitation, risk assessment and audit water safety plans; and also evaluates the existing paradigm, institutional and administrative framework for WSS services in urban areas to inform the development of a new management framework. Water policy implementation gaps are examined using a four-part capacity framework that includes institutional, technical/human, financial and social capacities. The framework for assessment of service level is informed by the human rights principles on water and sanitation provision that incorporates the human rights normative and cross-cutting criteria. Data for the study is collected from a literature review, semi-structured interviews with households and other stakeholders that include water service authorities and other institutions that were systematically selected. Additional data is collected from water sampling using the Compartment Bag Test (CBT), field observations across communities in Masvingo and Harare city councils with residential (suburb) categories used as units of analysis. Both descriptive and inferential and risk assessment. The Statistical Package for Social Sciences (IBM SPSS) version 26 is used to analyse quantitative data and a thematic approach used to analyse qualitative data. Themes are drawn phenomenon under study and data coded and put into categories based on the research aims. The study also identifies the elements of implementation of the ZNWP that constrain the provision of services in urban areas in the City of Masvingo and Harare and these are: financial, institutional, technical/human and social capacities. Other constraints that include political al meddling in city council duties by the central government, financial and technical/human resource capacity shortages that impact on urban local authorities (ULAs)’s capacity to deliver on WSS services are also considered in the study. The results show that financial capacity is needed to support the ZNWP programmes that include the provision of drinking water and recruitment of skilled staff among others. The notes the need for adequate political support to implement the ZNWP programmes, adequate financial support to be offered to ULAS by central government to assist in capital intensive programmes investments such as WSS service augmentation, and for transparency , communication, education and awareness to be enforced at all levels in ULAs. The study recommends that there be an urgent development of water safety plans including education on household water treatment and safe storage (HWTS), and for the establishment of an emergency response plan by ULAs to safeguard public health and protect the right to safe drinking water for all. Consequently, the study proposes a new institutional and administrative framework that includes modifications of the old framework to reduce or eliminate the identified gaps, incorporates an independent WSS regulator who must enforce regulation of WSS services, and consists of a component of urban WASH that should be overseen by the Ministry of Health and Child Welfare’s Department of Health in light of recent episodes of drinking water related diseases which are mostly the result of poor hygiene including poor storage drinking at household level. The new institutional and administrative framework should also ensure an active role of water users by including civil society organisations (CSOs) such as the rate payers’ associations as legal entities in the management of WSS services.Item Embargo Molecular characterization of entamoeba species and impact of host genetic polymorphisms on amoebiasis(2022-11-10) Davhana, Ndivhudzannyi Caroline; Samie, A.; Bessong, P. O.; ElBakri, A. M. K.Background: Enteric infections constitute a serious public health problem globally, especially in low and middle-income countries, particularly in areas of poor sanitation, low socio-economic conditions, inadequate water supply and poor hygiene practices. South Africa is one of the developing countries that has been significantly impacted by diarrheal infections, many of which are due to Entamoeba species. It is still not clear why some individuals once infected with E. histolytica develop clinical amoebiasis while others do not show any symptoms. It has been suggested that both the parasite and host factors play a significant role in the outcome of E. histolytica infection. However, this does not explain why only few infected individuals develop symptomatic diseases. This suggests that there are other factors to explain this transition. Tumor necrosis factor-α (TNF-α) is a multifunctional pro-inflammation cytokine which has been considered as one of the pathogenic factors for various diseases. Several studies have reported an association between TNF-α polymorphisms and inflammatory diseases. However, no study has been carried out on the association between polymorphisms in the promoter of the TNF-α gene and high risk of E. histolytica infections. Aim and objective of the study: The overall aim of the study was to determine the molecular characteristics of Entamoeba species in relation to the occurrence of diarrhea among children and determine the impact of host genetic polymorphism on the occurrence of amoebiasis. This aim was addressed by the following primary objectives: a. To investigate the prevalence, distribution and genetic diversity of Entamoeba species and other parasites circulating among children in the Northern part of South Africa. b. To investigate the genetic polymorphism of TNF-α gene promoter region in a cohort of children in Northern South Africa. c. To identify any association between TNF-α promoter gene polymorphism with diarrhea, vomiting, fever, acute lower respiratory infection, gender and malnutrition. d. To identify any potential association that may exist between TNF-α promoter gene polymorphism and parasitic infections. Brief methodology and results: This study was nested within the Madi project and Malnutrition and Enteric Diseases project (MAL-ED) South Africa and received approval from the Health, Safety and Research Ethics Committee of the University of Venda. The stool samples were from Madi project and the blood samples were from the MAL-ED project. A total of 394 stool samples were collected in selected household with children under the age of 5 years who were randomized to receive a silver-impregnated ceramic water, a silver-impregnated ceramic disk, a safe-storage water container, or no intervention, and were followed quarterly for two years from Dzimauli rural communities of South Africa in Limpopo province. All the stool samples were observed under a light microscope for the presence of intestinal parasites. In order to accurately detect Entamoeba species in all faecal samples, polymerase chain reaction (PCR) protocols were performed using genus-specific PCR primers based on small-subunit rRNA gene sequences for E. polecki, E. chattoni, E. dispar, E. histolytica, E. hartmanni and E. coli. Real-time PCR protocol was also used to detect and identify the specific Entamoeba species such as E. histolytica, E. dispar, E. bangladeshi and E. moshkovskii in order to gain information on their accurate prevalence, distribution and genetic diversity in the community. The present study reported an overall prevalence of intestinal parasites including Entamoeba species, Strongyloides stercoralis, Cystoisospora belli and Ascaris lumbricoides as determined by microscopy to be 140 (35.5%), 138 (35.0%), 114 (28.9%) and 78 (19.8%), respectively. The genus-specific PCR was positive for Entamoeba spp. in 140 (35.5%) samples. Real time PCR detected E. histolytica in about 4% of the samples while E. moshkovskii occurred in about 9% of the samples. Results identified by qPCR showed that children in silver-impregnated ceramic water filter group at month 12 had higher E. moshkovskii infection 6 (13.0%), while those in intervention group had lower E. moshkovskii infection 3 (6.8%). None of the participants were infected with Entamoeba bangladeshi. Sequence analysis showed a wide variety of Entamoeba species with E. coli and E. polecki appearing to be the most common organisms followed by E. moshkovskii and E. dispar. Further studies using next generation sequencing technologies are needed to understand the real importance of each of these organisms in the community in terms of the pathogenesis of amoebiasis (Chapter 3). TNF-α is a multi-functional pro-inflammatory cytokine and a primary mediator involved in the early phase of the cytokine cascade and plays an important role in the initiation and regulation of the immune response. The present study aimed to investigate the genetic polymorphism of tumor necrosis factor-α (TNF-α) gene promoter region in a cohort of children in Northern South Africa. A total of 199 blood samples were evaluated from children who were part of the MAL-ED study cohort. The TNF-α gene at positions ‑1031T/C and ‑308G/A were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay and confirmed by DNA sequencing. Out of these 199 participants, 94(47.2%) were males and 105(52.8%) were females. Of all the children, 23(11.6%) had low birth weight. A strong association was noted between the CC homozygous genotype at position -1031 and children with diarrhea (P=0.043, OR=4.167, 95% CT=0.942-18.43); whereas TC heterozygous genotype was significantly common in healthy children with no diarrhea (P=0.019, OR=0.446, 95% CT=0.226- 0.882). The T-allele was significantly common in children with diarrhea (P=0.043, OR=0.240, 95% CT=0.054-1.062). A strong association was also noted between the TT homozygous genotype at position -1031 and children with dehydration (P=0.014, 95% CI= 1.224-12.443). A strong association was noted between the GA heterozygous genotype at position -308 and children with diarrhea (P=0.040, OR=2.579, 95% CT=1.019-6.528); while AA homozygous genotype was significantly common in healthy children with no diarrhea (P=0.012, OR=0.3420, 95% CT=143-0.815). Heterozygous GA genotype was more common among healthy children with no dehydration and the result was statistically insignificant (P = 0.894, X2 = 0.018, OR = 1.095, 95% CT = 0.288-4.168); while a strong association was also noted between the heterozygous GA genotype at position -308 and children with vomiting (P=0.019, OR=2.694, 95% CT=1.160-6.256). The G allele was significantly common in children with vomiting (P=0.010, OR=2.816, 95% CT=1.263-6.279). Our study has for the first time revealed that the -1031(T/C) polymorphism of TNF-α promotor gene is associated with diarrhea, dehydration and acute lower respiratory infection among children at five years of age, while the -308(G/A) polymorphism was associated with diarrhea and vomiting among these children (Chapter 4). Intestinal parasitic diseases are common in developing countries including South Africa and have been documented to be the most common in children under the age of five. The present study aimed to identify any potential association that may exist between TNF-α promoter gene polymorphism and parasitic infections. A total of 199 blood samples was evaluated from children who were part of the MAL-ED study cohort. The DNA was used to investigate polymorphism in the promoter region of the TNF-α gene at position 1031T/C. The polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. The TC genotype at position -1031 was significantly higher in healthy controls children than in children who were infected with Entamoeba species (59.9% vs 29.4%, P = 0.015) and Entamoeba coli (59.1% vs 30.8%, P = 0.046), indicating that TC genotype may be protective against Entamoeba infections and Entamoeba coli infections. The CC genotype at position -1031 was more common among children with parasite and diarrhea and the results was statistically significant (P = 0.04). This study has revealed that the CC genotype may be is a risk factor for symptomatic parasitic infections, while the TC genotype might be protective of Entamoeba infections among children in Dzimauli community (Chapter 5). Overall conclusion: The present study demonstrated new data on the prevalence, distribution and genetic diversity of Entamoeba species and other parasites in Northern South Africa. Several Entamoeba species are circulating in the region and the importance of E. moshkovskii in the pathogenesis of amoebiasis seems to be underestimated. This study also revealed for the first time that the -1031(T/C) polymorphism of TNF-α promotor gene is associated with diarrhea and acute lower respiratory infection, Entamoeba species and Cyclospora infections among the children in Dzimauli community, while - 308(G/A) was associated with vomiting and overall illness. Information generated from this study will be useful for understanding the transmission, source of infection and clinical outcome of infection with parasitic infections. However, larger studies need to be conducted in order to confirm these findings.Item Open Access Molecular characterization of human sapoviruses circulation in the rural communities of Limpopo Province, South Africa(2022-11-10) Magwalivha, Mpho; Potgieter, N.; Traore-Hoffman, A. N.Background: Viral diarrhoea is a common cause of mortality among children less than five years of age in developing countries. Sapovirus (SV), one of the enteric viruses has been reported to be associated with viral diarrhoea worldwide. Reported studies on SVs in selected provinces of South Africa (SA) have been published based on the patients admitted in the hospitals located in the urban areas. There is a need for continuous epidemiological studies of SVs from the rural based regions within SA, especially from outpatients reporting in rural health care centers. Objective: To determine the genotypes, characterize, and analyze a capsid protein of the detected human Sapovirus strains associated with diarrhoea in children less than 5 years of age from rural communities in SA, and also compare the detected SV strains in this study with other strains reported elsewhere around the world. Method: A review article on the prevalence of human SV in developing countries was compiled to support the rationale of this study. To find the SV genotypes, characterizing, and comparing them with previously reported SV strains, an investigation on the “Prevalence and genetic characterisation of human sapovirus from children in the rural areas of Vhembe district” was conducted. A total of 284 stool samples were collected from children under 5 years of age suffering with diarrhoea (n=228) and without diarrhoea (n=56). Samples were screened for SV using real-time PCR. Sapovirus positive samples were further analysed for genogrouping by a One-Step Ahead RT- ix PCR, and SV Strains were genotyped using Sanger sequencing. A polyprotein (partial capsid protein) was successfully amplified using One-Step RT-PCR from 25% (10/40) positive samples, and further sequenced using Sanger method. Results: From a review report, 6.5% prevalence rate for SV in the low and middle income countries was determined, with significance difference of SV prevalent rate seen between low income and middle income countries. This study reports 14.1% (40/284) SV detection from stool samples [16.7% (38/228) of diarrhoeal and 3.6% (2/56) of non-diarrhoeal samples]. Genogroup-I was found as the most prevalent strain comprising 68.75% (11/16), followed by SV-GII 18.75% (3/16), and SV-GIV 6.25% (1/16), with GI and GII detected in 6.25% (1/16) of the sample. Significant correlation between SV positive cases and water sources was noted (Chapter 4). A partial VP1 was successfully sequenced from 10/16 amplicons, and results showed genotype GI.1 to be the most prevalent (60%; 6/10), followed by 20% of SV-GII.1, and 10% of each SV-GI.3 and SV-GII.3 (Chapter 5). The relatedness of strains detected from non-human host with the detected strains from this study was noted with a concern (Chapters 4 and 5). Conclusion: The presence of SV, and substantial evidence of SV associated with diarrhoeal disease in low income regions was determined. This study defined human SV strains in rural communities from Vhembe district, and therefore outpatients in rural settings are possibly at a risk of the burden of diarrhoeal disease triggered by enteric-viruses among other pathogens. However, reports on SV as an emerging diarrhoeal causative agents in the developing regions are limited. Investigations on the analysis and surveillance of human SV strains in rural settings (at a community or household level) is essential to assess burden of diseases.Item Open Access Identification of acyl transferases responsible for the diverse hydroxycinnamoyl chemical space in bidens pilosa(2022-11-10) Mathatha, Khuliso; Madala, N. E.For decades, plants have been the backbone of complicated traditional herbal medicine system. Plants have been used by people and animals as a source of nutrients as well as medicine. Production of secondary metabolites by these plants is a characteristic that makes them attractive to both animals and humans. In plants, secondary metabolites play a role in defence mechanism and assist the plant to adapt to their immediate environment. Secondary metabolites are known to possess anti-diabetic, anti-malaria, anti-inflammatory and alleviate complications associated with obesity and cardiovascular diseases. Plants from the Asteraceae family are known to contain metabolites with nutraceutical properties. Plants such as Helianthus annuus, Lactuca sativa, Chicorium intybus and Bidens pilosa are some of the edible examples within the Asteraceae family known to exhibit interesting nutraceutical properties. B. pilosa adapt to almost every environmental condition, which makes it to be found in all parts of the world. As such, this plant has been used to manage and treat illnesses affecting humankind. Unique to this plant is the existence of large contingency of structurally diverse chlorogenic acids. B. pilosa is known to produce different structural hierarchies of chlorogenic acids (i.e., mono-, di-, tri- acyls). The biosynthetic pathway to produce the diverse array of chlorogenic acids in B. pilosa is not yet elucidated. It is known from other plants like Helianthus annuus that the production of chlorogenic acids is coded by hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase gene (HQT) and hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase gene (HCT). Apart from the chlorogenic acids (quinic acid acyls), B. pilosa is known to also produce acyls of tartaric acid, also characterised by existence of structurally diverse isomers thereof. From other plants, the tartaric acid esters are coded by the hydroxycinnamoyl-CoA: tartaric hydroxycinnamoyl transferase (HTT) gene and, surprisingly, the gene encoding the tartaric acid esters/acyls from B. pilosa is also not known. It is therefore imperative that a study aimed at establishing/identification of the gene elements which are responsible for the diversification of chlorogenic acids and related compounds (such as tartaric acid acyls) in B. pilosa is conducted. To achieve this, a Single Molecule Real Time (SMRT) sequencing approach was used to establish the full-length gene transcripts, in attempt to identify the acyl transferase Executive summary xi responsible for chlorogenic acids production in B. pilosa. The SMRT sequencing technique has brought a lot of improvement from the Sanger and Next generation sequencing such as generation of long reads which overcomes the challenges of sequence assembly synonymous with the short reads achieved by the former two sequencing approaches. Moreover, this technique allows detection of isoforms of a specific gene, caused by either inherited genetic code or alternative splicing events. From the SMRT sequencing results, three HQT genes and one HCT gene responsible for production of wide array of chlorogenic acids and two HTT genes responsible for production of tartaric acid esters were identified through series of bioinformatics analyses of the sequences obtained through SMRT sequencing. All the identified genes contained the conserved regions that are found in already published acyltransferases, with the highly conserved DFGWG motif present in all transcripts identified herein. The second motif, the HXXXD motif showed a single amino acid variation from gene to gene, with HQT1 and HQT2 showing HTLSD motif and HQT3 having HTLAD motif, all of which are synonymous with the plants from the Asteraceae family. In HTT genes, the second motif identified in B. pilosa has never been recorded in literature. HTT1 and HTT2 showed to have HRVLD and HRVAD motif respectively. From these sequences, the open reading frames (ORFs) were computed, and these sequences can be used to design primers that can be used to amplify these genes in the future. Through multi sequence alignment and phylogenetic trees, the identified genes were also found to have similarities with the genes of other plants from the Asteraceae family. In conclusion, the SMRT sequencing approach enabled identification of acyltransferases genes that plays a role in the biosynthesis of CGAs in B. pilosa. Bioinformatics tools were shown to be sufficient to annotate and characterise these genes. Through LC-MS analyses of randomly collected B. pilosa plants, the CGAs content of this plant were revisited, and these plants were found to produce structurally diverse CGAs compounds, suggesting that the identified genes are functional. Future studies should aim to clone these transcripts in plant systems that do not produce CGAs in attempt to enhance their nutraceutical attributes.