Thesis (Ph. D. (Microbiology)) --University of Limpopo, 2019 / Tuberculosis (TB) is one of the top ten diseases that causes morbidity and mortality worldwide. Although TB is curable, the main problem currently with TB is development resistance to the current chemotherapy. Medicinal plants, as a source of drugs, have been found to cause less or no resistance. Medicinal plants are studied and considered for their efficacy and safety because they possess bioactive compounds with various biological activities. The aim of the study was to isolate and characterise bioactive compounds from selected seven plant species [A. dimidiata (LNBG 1969/46), A. afra (LNBG 2010/27), Z. capense (LNBG 1969/100), C. herorense (LNBG 1977/71), L. javanica (LNBG 1969/460), E. camaldulensis and C. lemon (UNIN 12330)] with activity against Mycobacterium smegmatis, Multi- drug resistant tuberculosis starain and H37Rv Mycobacterium tuberculosis strain. It was also imperative to determine whether crude extracts, sub- fractions of the extracts and the isolated bioactive compounds are cytotoxic or not. Leaves of the seven selected plants were collected from South African National Botanical Institute (SANBI) at Nelspruit, Mpumalanga Province, South Africa. The leaves were dried and milled to fine powder. The leaves of each plant were extracted using solvents of varying polarity (i.e. hexane, dichloromethane, acetone and methanol). Phytochemical screening was done using Thin Layer Chromatography (TLC) developed in three mobile phases varying in polarity and then sprayed with vanillin sulphuric acid in methanol heated at 110oC for optimal colour development. Qualitative antioxidant activity was determined by using 1,2- diphenylpicryl hydrazyl (DPPH) assay on TLC plates. Antimycobacterial activity for all the plant extracts was done using bioautography assay in qualitative analysis of the active compounds and for quantitative analysis, the microplate dilution assay was used. The plants which showed better activity (C. lemon, C. hereroense and A. dimidiata) with the microplate dilution assay and bioautography were further subjected to solvent- solvent fractionation as the first step towards isolation of bioactive compounds. Synergistic, additive, indifferent and antagonistic effects of the crude extracts combinations of the three selected plants was determined. The combinations where A. dimdiata was also part of the combinations frequently showed synergistic effect. On the other hand, with the combinations of C. hereroense and A. dimdidata (CH-AD) there was no antagonistic effect observed. The combinations of crude extracts of C. lemon and A. dimidiata all showed synergistic effect, except for only three combinations. Based on the synergistic effect observed and the bioactivity on the bioautography and microplate dilution assay of the sub- fractions, A. dimidiata was chosen for further analysis for antimycobacterial activity using the MDR- TB strain and M. tuberculosis H37Rv strain. The sub- fractions of A. dimidiata with the most activity were hexane and butanol. Hexane and butanol fractions both showed good MIC activity against the TB isolated M. tuberculosis field strain and H37Rv strain of 0.47 and 031 mg/ml, respectively. Butanol fraction was further taken for isolation using open colum chromatography doing bioassay guided isolation. The isolated compounds, together with the crude were tested for their biological activity using MTT assay to determine their cytotoxicity and antimycobacterial activity assay to confirm their activity against M. smegmatis and M. tuberculosis. Cytotoxicity assay showed that the crude extracts of A. dimidiate were toxic against the Vero kidney cells and the subfractions (i.e. butanol and hexane) became moderate to non-toxic and one compound (oleanolic acid) from the butanol sub-fraction was non- cytotoxic. This indicates that the isolation of the crude extracts tends to become non- toxic to the cells. The study suggests the use of pure compounds to fight against TB as compared to crude extracts since they are both bioactive and non- cytotoxic. Crude extracts combinations were effective in killing Mycobacterium as compared to single crude extracts. The present study recommends the use of A. dimidiata plant leaves crude extracts combinations as they mostly exhibit synergistic effect. Furthermore, Mycobacterium and also contain non- cytotoxic antimycobacterial compound (oleanolic acid). The study serves as a scientific proof for the use of this plant in traditional medicine for TB treatment.

Mycobacterium tuberculosis and Human Immuno-Deficiency Virus (HIV) have a high prevalence in South Africa. The development and spread of drug resistant tuberculosis is a serious problem which is exacerbated by tuberculosis (TB) co-infection in HIV patients. Traditional medicinal plants like Annona muricata and Artemisia afra are used for respiratory ailments and antiviral therapies respectively. The aim of this study was to evaluate Annona muricata (ethanolic extract) and Artemisia afra (ethanolic and aqueous extracts) for inhibitory activities against M. tuberculosis and HIV. In vitro bioassays for anti-TB activity included: microplate alamar blue assay (MABA), flow cytometry and ρ-iodonitrotetrazolium chloride assays while anti-HIV activity was determined using an HIV-1 reverse transcriptase colorimetric ELISA kit and an HIV-1 integrase colorimetric immunoassay. Cytotoxicity of plant extracts were assessed by the MTT assay on Chang Liver and HepG2 cells. Potential synergistic effects were determined using the basis of Combination Index. Potential interactions of plant extracts with drug metabolic pathways were evaluated with the Glutathione-S-Transferase assay kit as well as the CYP3A4 assay kit. A. muricata ethanolic extract exhibited anti-TB activity with MIC 125 μg/mL. MABA was shown to be the most sensitive and effective method for the detection of anti-TB activity. Artemisia afra aqueous extract showed HIV-1 reverse transcriptase inhibition exhibiting ˃85 percent inhibition at 1 mg/mL while the ethanolic extracts of A. afra and A. muricata showed inhibition of HIV-1 integrase activity at ˃86.8 percent and ˃88.54 percent respectively at concentrations >0.5 - 4 mg/mL. The aqueous extract of A. afra displayed inhibition of HIV-1 integrase ˃52.16 percent at 0.5 mg/mL increasing to 72.89 percent at 4 mg/ml of the extract. A. muricata was cytotoxic at an IC50 of 30 μg/mL and 77 μg/mL on Chang Liver and HepG2 cells respectively, whilst A. afra aqueous and ethanol extracts were not cytotoxic to both cell lines. The ethanolic extract of A. muricata showed both antagonistic and synergistic properties at various IC values, when used in conjunction with rifampicin. A. afra ethanolic extract interrupted GST activity while aqueous extracts of A. afra and A. muricata had a slight effect. All extracts interrupted CYP3A4 activity, however the ethanolic extracts of A. muricata and A. afra showed greater inhibition than the aqueous extract of A. afra. These extracts should be investigated further as they could be an important source of compounds for treatment of M. tuberculosis and HIV respectively.

Thesis (M. A. (Communication Studies)) -- University if Limpopo, 2021 / This study was prompted by the number of people who die daily from tuberculosis (TB) in the study area. The study investigated the perceptions of Mokopane residents, and their level of understanding and knowledge of the disease, tuberculosis (TB). The aim of the study was to profile people’s perceptions of health beliefs, the causes of the increase and spread of TB and its prevention and their knowledge about TB awareness campaigns in the Mokopane area. Data was collected by means of both qualitative and quantitative methods. Ten (10) medical doctors who operate private practices in the Mokopane area were interviewed. Data was collected from four hundred and one (401) participants from both Sandsloot and Tshamahansi villages outside Mokopane. The major findings of this study showed that firstly residents have a firm belief that traditional methods cure TB; secondly, they are largely ignorant of TB, its prevention, and consequences; and lastly that there is a lack of TB related education.

Thesis (M.Sc.(Microbiology)) -- University of Limpopo, 2022 / This study aimed to investigate antimycobacterial and cytotoxic compounds from Cassia petersiana. Cassia petersiana was selected for the current study based on its traditional use for treating tuberculosis (TB) symptoms. Extraction is an important step in the use of medicinal plants; hence, solvents of varying polarity were employed to extract a wide range of compounds where chloroform was the best extractant (67 mg). As there is no relation between the amount of plant material extracted and the bioactivity of the extracts, standard tests were used to determine the presence of different phytochemical constituents from Cassia petersiana and the total phenolic, flavonoid, and tannin contents were quantified using colorimetric assays. It was revealed that all the tested phytochemical constituents were present, and it was proven that phenolic compounds were the most abundant, followed by the tannins, while the flavonoids were the least among the common phytochemical constituents quantified. The phytochemical compounds were further profiled on thin-layer chromatography (TLC) and developed in BEA, CEF, and EMW solvent systems. Colourful compounds which indicated diverse phytochemicals were visualised with both vanillin-sulphuric acid and ultraviolet light on the phytochemical chromatograms and good separation of the compounds was from the BEA solvent system. The qualitative and quantitative antioxidant activity and antimycobacterial activity assays were used to evaluate the extracts from Cassia petersiana. Minimal antioxidant activity was observed on the qualitative antioxidant activity profile. These findings correlated with the minimal quantity of antioxidants from extracts of Cassia petersiana from the quantitative antioxidant assays; ferric reducing power and DPPH scavenging activity assays. Cassia petersiana extracts had bioactivity against Mycobacterium smegmatis as indicated by the lowest MIC value. The cell viability effects of the acetone crude extract from Cassia petersiana were evaluated against the tryptophan hydroxylase-1 (TPH-1) macrophage cells. Large scale extraction procedure was employed to extract a sufficient amount of plant material in preparation for the isolation of the bioactive compound. Bioassay-guided fractionation combined with column chromatography and TLC were used to isolate and purify the bioactive compound from the n-hexane extract of Cassia petersiana. The purified isolated compound was elucidated as β-sitosterol, which showed remarkable bioactivity against Mycobacterium smegmatis only on the TLC-bioautographic assay, while the quantitative antimycobacterial activity was higher xx with the MIC value of 2.5 mg/mL. Although β-sitosterol is known as a good antioxidant, it showed no antioxidant activity on the qualitative antioxidant activity assay. Therefore, further studies, including in vivo assay, are recommended on the isolated compound to evaluate its biological activities before consideration of its use in the development of alternative drugs.

Thesis (MPH.) -- University of Limpopo, 2017. / The present study presents data about the prevalence and determinants of sputum smear non-conversion in smear positive tuberculosis patients. Despite the intervention by the Sekhukhune District Department of Health through continual training and workshops of professional nurses in respect of the NTCP, there are still more challenges observed in terms of TB management. Aim: To investigate the prevalence and determinants of sputum smear non-conversion in smear positive PTB patients after intensive phase of treatment. Method: Quantitative, descriptive retrospective study of TB records was conducted. Data collection was done by extracting data from ETR.net and exporting it to excel. Data cleaning was done before analysis. Data analysis was done using the computer Statistical Package Software for Social research (SPSS) volume 23.1. Findings: 834 TB patients’ records were extracted from the ETR.net database. 34% of records were available at 2 – months; 57% of the patients were males; also, 81% of the patients were diagnosed/treated at PHC facilities; 52% of the patients were HIV positive; 69% percent of the patients who were smear positive grading p+++ failed to convert after two months. In the univariate logistic regression patients with age 20 – 29 were observed to be 4.9 times likely (O.R. = 4.97) to be sputum positive (P = 0.142).Sputum grade 3(p+++) at the time of diagnosis was found to be significantly associated (P = 0.031) with sputum non – conversion after intensive phase of treatment. Conclusion: Two month sputum smear non-conversion is associated with pre-treatment sputum smear grading.

Thesis (MPH.) -- University of Limpopo, 2019 / Background: Tuberculosis still continues to be a global public health problem and leads to many deaths. In an effective TB control strategy, TB patients are allocated to community care workers who provide care to these patients in their homes. It is important to understand the experiences of community care workers in order to strengthen TB control in the country. Objective(s): The purpose of this study was to explore lived experiences of community care workers of supporting patients taking Tuberculosis treatment. Methods: The design of the study was phenomenological, exploratory, descriptive, and contextual. The study site was Hlogotlou area in Limpopo Province. The target population was all community care workers supporting patients on Tuberculosis treatment. Purposive sampling was used with a sample of 13 participants, which was determined by the saturation of data. Semi-structured interviews were conducted using an interview guide and all sessions were audio recorded. The data were analysed using Interpretative Phenomenological Analysis. Results: The results highlighted certain challenges met by community care workers. Patients thought that community care workers are there to kill them with treatment, they had mood swings during treatment and this caused them to use vulgar words and become aggressive to their community care workers. Most community care workers did not have enough information about Tuberculosis. This made it difficult for them to support patients on tuberculosis treatment. Conclusions: A good relationship with patients enhances treatment compliance. The researcher recommends that intensive training about tuberculosis should be provided to community care workers.

The purpose of the study was to develop best practice guidelines to prevent permanent hearing loss associated with the management of multi-drug resistant tuberculosis (MDR-TB) through raised awareness and monitoring. The Human Immunodeficiency Virus (HIV) and MDR-TB are global public health problems requiring urgent scale-up of treatment services. Irreversible sensorineural hearing loss (SNHL) is one of the adverse drug reactions of the current World Health Organization (WHO) recommended MDR-TB chemotherapy fuelling another public health problem, that disabling hearing loss, which is the second highest contributor of Years Lived with Disability (YLD) according to the World Health Report (2003). Expansion of MDR-TB treatment threatens to increase incidence of SNHL unless there is urgent implementation of intervention towards preservation of hearing for patients on treatment. This empirical study determined and documented the incidence of SNHL in HIV positive and HIV negative patients on MDR-TB treatment, the risk factors for SNHL, from the time treatment initiation to SNHL. Based on the findings, developed and improved the understanding of best practice guidelines for monitoring and prevention of MDR-TB treatment-related SNHL. The empirical study recruited a cohort of 173 patients with normal hearing status, after diagnosis with MDR-TB and enrolled on MDR-TB therapy over thirteen month period. Patients in the cohort received monthly hearing sensitivity testing during the intensive MDR-TB therapy when injectable aminoglycoside antibiotics are part of the treatment regimen. The three study endpoints included completion of the eight-month intensive treatment phase without developing hearing loss, development incident hearing loss or loss to follow up. Data was analysed using STATA statistical software and summarised using frequencies, means, proportions, and rates. The study documented incidence of SNHL, time to hearing loss and risk factors for hearing loss. Recommendations to prevent and monitor hearing loss are made based on the the study findings. / Health Studies / D. Litt. et Phil. (Health Studies)