A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science Johannesburg, August 2012 / It is known that infectious agents elicit different responses in different individuals which strengthens the view that susceptibility and resistance to infectious diseases has a genetic component. These differences in susceptibility to disease can be observed in populations. APOBEC3G is a member of the cytidine deaminase gene family located on chromosome 22. It is crucial in non-permissive cells as it functions as part of the innate immunity system and is an inhibitor of the HIV-1 accessory protein vif. The goal of the study was to develop genotyping assays and estimate allele frequencies. Thus, genetic variation within APOBEC3G was identified and characterized in black South Africans. Indirect genotyping assays were designed to amplify regions within the upstream non-coding region, and in exon 4 of the coding region of the gene. Selected polymorphisms were then genotyped using allele-specific PCR, RFLP-PCR and Pyrosequencing™ assays. Reanalysis of sequence data from 2003 showed numerous SNPs were well represented. Comparison of sequence data at various SNPs showed that allele frequencies were similar to frequencies in other African populations. The only sequenced SNP that deviated from the frequencies in Ensembl was -590. Thus the sequencing was a useful tool for detection of variation. ASA proved to be the least reliable genotyping technique as the minor allele frequency of -571 (0.59) deviated from the published frequency of 0.894 in Africans. RFLP analysis proved more reliable for genotyping -571 and H186R. The minor allele frequency was estimated to be 0.84 and 0.32 for -571 and H186R respectively. The frequency of H186R is similar to published data from An et al (2004) and Reddy et al (2010). If SNPs are in LD they occur together on the same haplotype more often than by chance. Usually SNPs that are in LD are in close proximity. However our data suggests -571 and H186R SNPs which are 5kb apart are not in LD. A LD map of chromosome 22 shows highly variable pattern of LD (Dawson et al, 2002). Widespread regions of nearly complete LD up to 804 kb in length are intermingled with regions of little or uundetectable LD. Haplotype analysis showed the most frequent haplotype was GA. This was the most frequent haplotype when the sample types were subdivided according to spoken language. in comparison to studies from An et al, (2004) D’ of the two SNPs was estimated at 0.967. The linkage disequilibrium (LD) revealed a non-independence of allele segregation because the loci analyzed were strongly linked in the Apobec 3 G gene. The data are consistent with greater genetic diversity of African populations and can form the basis for further evaluation of the role of variation in this gene in response to HIV.

The socio-economic impact of HIV/AIDS on South Africa has resulted in lower gross domestic product, loss of skills in key sectors such as education, and increased health-care costs in providing access to treatment. Currently active pharmaceutical ingredients (API’s) such as stavudine (d4T) and azidothymidine (AZT) are imported from India and China, while formulation is conducted locally. A strategy was initiated between CSIR Biosciences and LIFElab under the auspices of Arvir Technologies to investigate the feasibility of local antiretroviral manufacture (d4T and AZT) or the manufacture of a key intermediate such as β- thymidine (dT). Several advantages associated with successful implementation of this strategy include ensuring a local supply of API’s, thus reducing reliance on procurement from foreign sources and reducing the effect of foreign exchange rate fluctuations on providing cost effective access to treatment. A local supply source would also reduce the imports and thus aid the balance of payments deficit, and in addition to this, provide stimulus in the local pharmaceutical manufacturing industry (which has been in decline for several decades), resulting in increased skills and employment opportunities. This thesis describes the development of a superior chemo-enzymatic process for the production of β-thymidine (72 percent yield, prior to isolation), a key intermediate in the preparation of anti-retrovirals. Alternative processes based purely on chemical or bioprocess transformations to prepare either 5-methyluridine (5-MU) or dT suffer from several disadvantages: lengthy transformations due to protection/deprotection strategies, low selectivties and product yields (30 percent in the chemical process) and isolation of the product from dilute process streams requiring the use of large uneconomical reactors (bioprocesss). This contributes significantly to the cost of d4T and AZT manufacture. Our novel chemoenzymatic process comprises of a biocatalytic reaction for the production of 5-MU, with subsequent chemical transformation into dT (3 steps) negating and circumventing the limitations of the chemical or bioprocess routes. During the course of this project development, the β-thymidine selling price declined from 175 $/kg (2005) to 100 $/kg (2008). However, the process described in this work is still competitive based on the current β- thymidine selling price of 100 $/kg. The process economics show that with further optimization and increasing the isolated dT yield from 70 percent to 90 percent, the variable cost decreases from 136 $/kg to 110 $/kg. The increase in isolated yield is highly probable, based on solubility data of β-thymidine. The decrease in β-thymidine selling price and technological improvement in dT manufacture should translate into lower API manufacture costs and more cost effective access to treatment. Our novel biocatalytic process producing 5-MU uses a coupled enzyme system employing PNP, Purine Nucleoside Phosphorylase and PyNP, Pyrimidine Nucleoside Phosphorylase. The overall transglycosylation reaction may be decoupled into the phosphorolysis reaction (PNP) and synthesis reaction (PyNP). During the phosphorolysis reaction, guanosine is converted into guanine and ribose-1-phosphate (R-1-P) in the presence of PNP enzyme. The reaction intermediate R-1-P is then coupled to thymine in the presence of PyNP enzyme during the synthesis reaction, producing 5-MU. The process was scaled up from lab-scale to bench-scale (10 - 20 L) and demonstrated to be robust and reproducible. This is evident from the average guanosine conversion (94.7 percent ± 2.03) and 5-MU yield (88.2 percent ± 6.21) and mole balance (104 percent ± 7.61) which were obtained at bench-scale (3 replicates, 10 L). The reaction was carried out at reactor productivities of between 7 – 11 g.L-1.h-1. The integration of the biocatalytic process and chemical processes was successfully carried out, showing that 5-MU produced using our novel biocatalytic process behaved similarly to commercially available 5- MU (ex. Dayang Chemicals, China). A PCT patent application (Ref. No. P44422PC01) on this chemo-enzymatic process has been filed and currently public private partnerships are being explored through Arvir Technologies to evaluate and validate this technology at one ton scale.

This study aimed to explore and describe the quality of life of adolescents living with early childhood HIV infection on Highly Active Antiretroviral Therapy (HAART) in Port Elizabeth. The advent of HAART has resulted in HIV being managed as a chronic illness, instead of the fatal disease that it once was. Children born with HIV can now live longer lives, progressing to adolescence and beyond. Chronic illness is known to impact one’s quality of life, so does adolescent development. A convenient sample of 31 adolescents was used in this study, with an exploratorydescriptive research design. The data was gathered using a cross cultural structured questionnaire developed by the World Health Organization, as well as through individual interviews. The data was then analysed by means of descriptive statistics and thematic content analysis. The results identified and presented the quality of life issues that are specific to this population. The results indicate that HIV as a chronic illness does impact the quality of life of adolescents. The adolescents living with early childhood HIV-infection on HAART in this study were very satisfied with their perceptions of their overall quality of life and general health perceptions. They were least satisfied in the Spirituality/Religion/Personal Beliefs and Social Relationships domains; and were most satisfied in the Level of Independence and the Psychological domains. There is a need for the development of (medical and psychosocial) services that can focus on adolescents as a special population with specific developmental needs in order to improve their treatment outcomes and quality of life.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 28 May 2018. / Since their discovery, protease inhibitors continue to be an essential component of antiretroviral treatment for human immunodeficiency virus type 1 (HIV-1). However, the development of resistance to protease inhibitors remains one of the most significant challenges in the fight for sustained viral suppression in those infected with HIV-1. Studies show that specific mutations arising within the HIV-1 gag and protease genes can lead to the development of resistance. In this research, a South African HIV-1 subtype C Gag-protease variant (W1201i) was investigated. This variant was considered due to the presence of a mutation and insertion (N37T↑V), located within the hinge region of the protease enzyme. Moreover, the variant displayed the following polymorphisms: Q7K, I13V, G16E, M36T, D60E, Q61E, I62V and M89L. Genotyping of W1201i Gag revealed a previously unreported MSQAG insertion between the CA/p2 and p2/NC cleavage sites. Additionally, a mutation and insertion (I372L↑M), and multiple polymorphisms (S369N, S371N, I373M and G377S) were discovered within the p2/NC cleavage site. Single-cycle phenotypic assays were performed to determine the drug susceptibility and replication capacity of the variant. The results show that the mutations present in the N37T↑V protease conferred a replicative advantage and reduced susceptibility to lopinavir, atazanavir and darunavir. Interestingly, the mutations in W1201i Gag were found to modulate both replication capacity and protease inhibitor susceptibility. In silico studies were performed to understand the physical basis for the observed variations. Molecular dynamics simulations showed that the N37T↑V protease displayed altered dynamics around the hinge and flap region and highlighted the amino acids responsible for the observed fluctuations. Furthermore, induced fit docking experiments showed that the variant bound the iv protease inhibitors with fewer favourable chemical interactions than the wild-type protease. Collectively, these data elucidate the biophysical basis for the selection of hinge region mutations and insertions by the HI virus and show that protease, as well as Gag, needs to be evaluated during resistance testing. / EM2018

Thesis presented in partial fulfilment for the degree of Master of Management (in the field of Public Sector Monitoring and Evaluation) to the Faculty of Commerce, Law, and Management, University of the Witwatersrand 2016 / The purpose of this research was to assess outcomes of the antiretroviral treatment intervention in Mankweng. The antiretroviral treatment intervention was conceived with the aim to reduce HIV transmission through viral load suppression. Literature has shown that viral load is used as a tool to measure the performance of the intervention and studies on viral load outcomes in rural settings of Limpopo are limited. For this reason, the research was focused on viral load suppression with the aim of (1) determining the proportion of adults with viral load suppression among people taking antiretroviral treatment for 12 months, and (2) identifying factors associated with failure to achieve viral load suppression among people on antiretroviral treatment for 12 months. Binomial logistic regression model was used to identify factors associated with failure to suppress viral load. This study used the theory of change to interpret its findings as well as theories of behavioural planning and self-regulation models to understand the logic that underpins the theory of change. The findings revealed that the majority (78%) of adults achieved viral load suppression, and a quarter of the individuals failed to suppress the viral load. There was no significant difference in baseline characteristics between people who achieved viral load suppression and those who did not. In addition, males, with a low CD4 and opportunistic illnesses when in the primary disease stages were more likely to fail to suppress the viral load. Moreover, people who were initiated on a 3-pill containing treatment and do not have social support were also likely to fail to achieve viral load suppression. Although the study showed a trend of likelihood towards failure to achieve viral load suppression, the estimates were not statistically significant. Theoretical arguments from this study suggest that failure to achieve viral load was attributed to poor adherence to treatment. This is supported by the logic that underpins the theory of change, in which the assumption of adherence was possibly violated. However, the results chain framework highlighted that the implementation of the intervention was effective as it led to good outcomes and an effective intervention. Drawing all this together, the study highlights the need for intensified adherence counseling during treatment in order to improve the performance of the intervention. Author: Ramatsobane Johanna Ledwaba, Thesis Title: Outcomes of the antiretroviral treatment intervention in Mankweng, Johannesburg, March 2016 / GR2018

Submitted in complete fulfilment for the Degree of Doctorate of Technology (Biotechnology)--Durban University of Technology, 2013. / South Africa has the largest number of people infected with HIV/AIDS. It also has more than 30 000 species of plants and many of these have a long tradition of medicinal use. It is highly likely that the treatment for HIV will come from this traditional knowledge. The need for effective preventative and therapeutic agents for HIV remains an urgent global priority. The aim of this study was to screen selected South African medicinal plants for anti-HIV activity and to identify and characterise an active compound from a plant that can be used for HIV treatment. The aqueous and methanolic extracts of the roots, leaves, flowers and stems of thirty eight plant species (108 extracts) were screened for anti-HIV activity. The plants which had anti-HIV activity were further screened for anti-reverse transcriptase activity. Thirty-two extracts exhibited varying degrees of anti-HIV activity. Cleome monophylla, Dichrostachys cinerea and Leonotis leonurus aqueous leaf extracts had anti-HIV-1 reverse transcriptase activity. The aqueous extracts of D. cinerea showed the best anti-HIV activity with a Selectivity Index of 43.5 and significant anti-HIV-1 reverse transcriptase activity. Crude phytochemical screening of D. cinerea showed that it had tannins, saponins, flavonoids and alkaloids but did not contain any phlobatannins, terpenoids, steroids or phenols. D. cinerea displayed a high degree of free radical scavenging activity with an IC50 of 25 μg/ml, therefore the anti-HIV activity could be attributed to the flavonoids present in the plant. Bio-guided fractionation was used to isolate and purify the active compound from the D. cinerea extract. Compounds were isolated by thin layer chromatography and were tested for anti-HIV-1 and anti-reverse transcriptase activity. From these results the active compound was identified, and purified using preparative TLC. The active compound was characterised by High Performance Liquid Chromatography, Ultraviolet-visible spectrophotometry, and Ultra Performance liquid chromatography coupled to MS/MS. Structural elucidation was performed using Nuclear Magnetic Resonance. From these results, it was deduced that the compound isolated from D. cinerea was a catechin. In this study we show that the catechins present in D. cinerea are responsible for the anti- HIV-I activity and inhibits the reverse transcriptase activity which is a key factor in the progression of HIV. Potentially, these results can be used to develop a new drug for the treatment of HIV or as a cost effective therapeutic agent in treating HIV-infected individuals with oxidative stress. / National Research Foundation

Thesis (MFamMed)--Stellenbosch University, 2015. / ENGLISH SUMMARY: Background: Sub Saharan Africa is home to approximately two thirds of the world’s population of HIV positive individuals. In view of the socioeconomic challenges of the region governments have provided antiretroviral therapy free to improve chances of survival among patients. However, adherence to antiretroviral therapy is recognised as more important in predicting patient survival. Aim: The study aimed to describe the prevailing factors that influence adherence to antiretroviral therapy. Methods: The study design was carried out using three methods of data collection. Focus group discussions, semi structured interviews and a questionnaire format. Setting: This study has been conducted among adult patients attending the Dora Nginza Hospital Adult wellness clinic. Results: The methods highlighted factors that influence antiretroviral therapy revealing psychosocial factors – lack of family support, not wanting to take medicines in front of people outside the home; patient factors – co morbidities that disturb patient adherence to therapy, a lack of trust in the patient-care giver relationship, fear of the drug side effects; socioeconomic factors – patients’ inability to afford food or transport costs to clinic appointments. In the semi structured interviews, 25% of patients self reported on poor adherence while in the questionnaire 5% of patients reported poor adherence. In assessing the effect of adherence to therapy on CD4 count and viral load there was a general increase in CD 4 count and a drop in viral load indicating clinical improvement in patients on therapy. Recommendation: There is a need for clinicians developing a health relationship with patients to facilitate adherence. The interventions designed to help patients in adherence to therapy should involve the patients in question in the designing of these interventions. Conclusion: Adherence monitoring is an important aspect of managing patients on antiretroviral therapy. The factors highlighted are similar to findings of other studies in similar contexts i.e. resource poor settings. / AFRIKAANSE OPSOMMING: Opsomming nie beskikbaar.

M.A. (Public Management and Governance) / This study starts with an introduction and background, and this is followed by a statement of the research problem, research rationale, research objectives, research design, research methodology and the data collection methods. A list of acronyms will be provided, as well as an overview of chapters, conclusions, and acknowledgements. “Around 5.7 million in 2009 had contracted HIV and AIDS and in the preceding year 250,000 South Africans died due to HIV/AIDS related diseases. All age categories and gender are prone to contracting the disease and the resultant loss of life has had a devastating effect on the workforce. Death due to this disease has caused loss of income and family disintegration” (Sottile 2013:2). In 2010, some 280,000 South Africans died of HIV/AIDS. In the decade up to 2010, between 42% and 47% of all deaths among South Africans were HIV/AIDS-related deaths (Sottile 2013:2). Mlambo et al (2011:1) further say that “The Human Sciences Research Council estimates that 10.9% of all South Africans have HIV/AIDS. Additionally, the Central Intelligence Agency estimates that 310,000 individuals died in South Africa from HIV/AIDS in the year 2009”. A study in 2003 made a comparison on two scenarios, an HIV/AIDS scenario and a no-HIV/AIDS scenario and this was based on the annual growth rates between 2002 and 2015. The finding was that “real growth in GDP would be 0.6% lower than if there were no HIV/AIDS, but per-capita growth in GDP would be 0.9% higher. Growth in population would have been 1.5% lower, and growth of the labour force would be 1.2% lower, but the unemployment rate would be 0.9% lower as well” (Avert.Org 2010:2). Different HIV/AIDS prevention strategies are being implemented in South Africa. The focus in this study is on male medical circumcision (hereafter MMC), which is the removal of all or part of the foreskin on the penis. It can either be performed surgically in hospitals or at initiation schools. Initiation schools perform the procedure as a traditional rite of passage from boyhood to manhood, and this is referred to as traditional male circumcision (TMC). This practice has up till now been performed purely for traditional or religious purposes, but it can no longer be viewed only as such. It should, instead, also be seen as a measure to curb the spread of the HIV/AIDS pandemic. A number of observational studies (which will be referenced later) found that circumcised men had smaller chances of contracting HIV infection compared with uncircumcised men...

The introduction of antiretroviral drugs (ARVs) in 1996 transformed the treatment of HIV and AIDS, improving the quality and greatly prolonging the lives of many infected people. HIV (Human Immunodeficiency Virus) is the virus that is believed to cause AIDS. AIDS (Acquired Immune Deficiency Syndrome) is the collection of illnesses or symptoms that ultimately results in death. Antiretroviral (ARVs) drugs or Highly Active Antiretroviral Therapy (HAART) is the treatment that has been applied to combat the HI virus in a bid to slow down the progression of AIDS and ultimately prolong the life of the infected individual. The study aimed to explore and describe the factors contributing to adherence and non-adherence to ARVs in individuals on treatment. A sample of 81 individuals who have been on ARV and HAART treatment for six months or more was used. The methodology used was exploratory-descriptive and the data obtained was quantitative in nature. A biographical questionnaire and questionnaire with questions aimed at ascertaining the possible factors that contribute to individuals either adhering to or defaulting on their treatment, such as side effects and cost of treatment, was administered. The data obtained was analysed by means of descriptive statistics and frequency counts. The results of the study showed that the sample had a fairly high level of adherence. The factors that could undermine adherence were identified as lack of support, as familial and health provider support acts as a motivator to adhere; substance abuse as it can lead to forgetting to take treatment; unemployment and poverty, as these can lead to an inability to return for follow up clinic visits or failure to have food to take with the pills; and the type of treatment regimen whereby the more complex the treatment is the more likely it is that adherence will be difficult to maintain. Suggestions were made as to future research involving antiretroviral therapy (ART). Finally the limitations as well as the value of the research were outlined.

Thesis (MPhill)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The mining industry having been identified as a particularly vulnerable sector to the potentially devastating effects of HIV and AIDS, it became clear that proper management thereof is essential if we are to mitigate these. Two-hundred-and-seventy-nine of the 720 patient files from the mine‟s wellness clinic were reviewed for clinical appropriateness. These files were scrutinised to evaluate adherence to treatment guidelines. The review was conducted using the wellness clinic‟s treatment guidelines, adapted from the South African HIV Clinicians Society (SAHIVCS) 2008 guidelines asking the four questions: “when to start therapy”, “what therapy to start”, “co-existence of other illnesses”, “when to change therapy”. The guidelines were largely adhered to as far as starting the right type of therapy at the right time. “When to start therapy” was adhered to in 97.1 percent of the cases, “when to start” in 99.6 percent. However evaluation of “co-existence of other illnesses” came in at a rather low figure of 45.5 percent. Although the need for change of therapy was very low (2.5 percent), 57.5 percent of those who needed a change in therapy were prolonged on failing regimens for periods of more than three months. / AFRIKAANSE OPSOMMING: Die mynindustrie in Suid-Afrika is as 'n uiters kwesbare sektor geïdentifiseer en behoorlike bestuur van hierdie sektor is van kritieke belang in die suksesvolle bestuur van MIV/Vigs in Suid-Afrika. Die mediese inligting van 279 pasiënte is vir die doel van die studie ontleed en die mate waartoe pasiënte getrou hou by behandelingsriglyne is in die studie ge-evalueer. Die ontleding was gebasseer op die 2008 welwees kliniese riglyne van die South African HIV Clinicians Society (SAHIVCS). Die studie bevind dat pasiënte in 'n groot mate by die kliniese riglyne gehou het en dat die datum waarop met behandeling begin is in bykans 97% van die gevalle ooreenkomstig die riglyne was. Die grootste bron van kommer was die groot relatief groot aantal gevalle waar die invloed van ander siektetoestande nie behoorlik in ag geneem is nie en die MIV/Vigs medikasie nie dienooreenkomstig aangepas is nie. Voorstelle word in die studie gemaak vir aksies wat geneem behoort te word om 'n groter mate van voldoening aan die riglyne te bewerkstellig.