M.Tech.

The standard anti-retroviral drugs (ARVs) used for the treatment of HIV/AIDS have significant side effects resulting in a lack of adherence and the emergence of multidrug resistant viral strains. These drugs are also expensive, making it essential to investigate all alternatives to classical HIV/AIDS treatment. A wide variety of nonconventional medicines are used by patients for the treatment HIV and for symptoms associated with HIV. So long as they are safe and effective, traditional, complementary and alternative medicines (TCAMs) may be considered more advantageous for developing countries as they are relatively cheap, more accessible and widely accepted by local populations. The aim of this study was to determine the prevalence of TCAM use in HIV-positive patients, prior to, and during ARV therapy. The study was exploratory, cross sectional and observational in nature. Participants were selected via convenience sampling from the Nelson Mandela Bay Municipality, and included 244 HIV-positive patients, 29 health care professionals (HCPs) and 30 traditional, complementary and alternative practitioners (TCAMPs). A wide variety of TCAMs were used by the sample population. These medicines were more commonly used by non-ARV patients (36 percent) compared with ARV patients (22 percent). A significant statistical difference in TCAM use between the ARV and non- ARV population was found in relation to education, employment, period of status awareness, patient opinion of personal health and the reasons for TCAM use. Amongst the HCPs, 24 percent recommended TCAM use prior to ARVs, and 55 percent were aware of patients self-prescribing before and during ARV treatment. Amongst the TCAMPs, 90 percent provided a wide range of TCAMs for HIV, with some giving consideration to conventional management. TCAMs are commonly used by HIV-positive patients on ARVs, as well as by those not on ARVs. These medicines are also the preferred form of treatment for those not seeking conventional treatment. TCAMs are widely available and recommended by TCAMPs as well as some HCPs. Due to public health concerns, clinical trials of the widely used TCAMs are crucial in order to establish the safety and efficacy of these medicines in HIV.

HIV infection has risen to pandemic proportions. Interleukin-10 (IL-10), a potent antiinflammatory cytokine has been shown to enhance the establishment and persistence of chronic viral infections through inactivation of effector antiviral immune responses and it may also directly influence HIV-1 replication in cells of diverse lineages. IL-10 promoter polymorphisms have been shown to affect HIV-1 susceptibility and pathogenesis. However, the underlying mechanisms are poorly understood. We investigated the relationship between IL-10 promoter variants, plasma IL-10 levels, and markers of disease outcome in chronically HIV-1-infected individuals. To investigate the mechanistic role of IL-10 and its genetic variants on HIV pathogenesis, we studied markers of activation on B cells, CD4+ and CD8+ T cells, and assessed effects on CD4+ T cell proliferation with and without blockade of the IL- 10 pathway. We used Taqman genotyping assays to genotype three IL-10 promoter single nucleotide polymorphisms (SNPs) in our study cohort. Baseline plasma IL-10 levels were measured using Luminex technology for 112 individuals. Viral load, CD4+ T cell counts and cytotoxic T lymphocyte (CTL) immune responses were measured at baseline. The rate of CD4+ T cell decrease was calculated in 300 individuals with a median follow-up of 25 months. CD38, CD95, Ki67, IgG and PD-1, markers of activation or exhaustion were measured on B cells, and CD38, CD95, Ki67, HLA-DR and PD-1 were measured on CD4+ and CD8+ T cells in a subset of 63 individuals. CD4+ T cell proliferation was measured using Carboxyfluorescein succinimidyl ester (CFSE) assays, following IL-10 receptor blockade in a subset of 31 individuals. The IL-10 -1082G, -592A and -3575 variants were observed at frequencies of 0.3, 0.34 and 0.23 respectively, in our study cohort. Plasma IL-10 levels were significantly higher in the - 1082GG group than in the combined AA/AG group (p=0.0006). There was a significant association between the 592AA genotype and a greater breadth of CTL responses compared to the CC and CA (p= 0.002 and 0.004 respectively). The -592AA genotype associated significantly with an attenuated loss of CD4 cells (p= 0.0496), with -592AA having the least change in CD4 cells per year. The median expression of HLA-DR, a marker of T cell activation was significantly higher in the-1082AA group for CD8 cells (p= 0.047), and the - 592AA group for CD4 T cells (p= 0.01). The median expression of IgG on the surface of B cells was significantly higher in the -1082GG genotype and the -592CC genotype (p=0.0183 and 0.0659 respectively). Overall, IL-10 variants correlated with IL-10 expression and CD4 decline during chronic HIV-1 infection. IL-10 promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels, which in-turn, may affect the breadth of CTL responses. Furthermore, the increased expression of HLA-DR and PD-1 on CD8+ and CD4+ T cells, indicates that lower IL-10 levels are associated with increased immune activation and immune exhaustion. The increased expression of IgG on B cells, suggests that in a setting of lower IL-10, there is possibly a bias towards a Th2 immune response. These data suggest a significant role for IL-10 genetic variants and IL-10 in HIV pathogenesis. Further studies to determine whether and how the IL-10 pathway may be manipulated for therapeutic or vaccine strategies for HIV are warranted. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

Currently, the approved anti-HIV drugs on the market only target the three HIV enzymes: reverse transcriptase, protease and more recently, integrase. Due to the limited nature of the current therapy, it is possible that a multi-drug resistant virus can emerge. The main concerns in developing countries however, are the expense and availability of the drugs and because of this, it is essential to investigate all alternatives. Traditional medicine offers many advantages as compared to allopathic treatment in so far as being relatively cheaper, accessible and it is broadly accepted in the population groups of the developing countries. Little is known though, of the exact efficacy and toxicity of these remedies so it is vital that these possible leads be investigated thoroughly. For the purpose of this study, two plants, Sutherlandia frutescens and Lobostemon trigonum were studied to ascertain their potential anti-HIV activity. Sutherlandia has received international attention as a possible cheap herbal remedy to improve the health of AIDS sufferers. Anecdotal evidence from health workers claim that HIV- infected patients on Sutherlandia treatment have shown improved CD4 counts, decreased viral loads and a general improvement in well-being. Extracts were prepared from dried leaves and flowers in methanol, ethanol, acetone, methylene dichloride or distilled water. Sulphated polysaccharides have been described extensively in literature with regards to their anti-HIV activity, so as a form of dereplication; an ethanol precipitation was performed on the aqueous extracts to remove sulphated polysaccharides. A toxicity study was performed on all crude extracts using uninfected peripheral mononuclear blood cells (PBMCs) isolated from whole blood. To measure anti-HIV activity, HIV-infected PBMCs were cultured with each of the crude extracts and cell viability measured using the tetrazolium salt, XTT. HIV-infected CEM-NKR-CCR5 cells were also used and supernatant from the viral studies was tested for the HIV antigen p24. xii Results varied greatly between assays but with the inclusion of a point-scale system to evaluate the extracts it was clear that overall the organic extracts of the Sutherlandia flowers, especially the acetone extract (SFA), showed great anti-HIV potential. SFA in every case decreased p24 levels and in the toxicity study did not decrease cell proliferation. With the HIV-infected PBMCs SFA actually helped improve cell proliferation despite the infection. To determine the specific anti- HIV activity, all crude extracts were tested for inhibition of HIV-I reverse transcriptase, the glycohydrolase enzymes: a-glucosidase, ß-glucosidase, ßglucuronidase, HIV-I integrase and HIV-II protease. No significant inhibition was seen with these experiments except for the HIV-I RT assay. The aqueous extract of the Lobostemon leaves produced an inhibitor of HIV-RT with a very low IC50 value of 0.049mg/ml. Some inhibitory effect was lost with the removal of the sulphated polysaccharides and the addition of BSA to the assay, but still 64% inhibition of the HIVRT remained, which confirmed that the inhibitor could be something novel, and not of the polysaccharide or tannin compounds.

Acquired Immunodeficiency Syndrome (AIDS) is a human disease caused by the human immunodeficiency virus type 1 (HIV-1) and it is one of the biggest social, economic and health challenges in the world. The Joint United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO) estimated that between 33.4 to 46.0 million people around the world were living with HIV/AIDS in December 2005 and the highest estimates are in the Sub-Saharan Africa (around 25 million). In more developed countries a combined antiretroviral therapy called highly active antiretroviral therapy (HAART) is used, which results in reduced progression to AIDS in most patients. Despite the beneficial effects of HAART, significant side effects are experienced by treated patients. In addition, most infected people live in countries where the treatment is very expensive or, in many cases, not available at all. These people therefore rely on medicinal plants for health care. In this study, aqueous extracts from Sutherlandia frutescens and Lobostemon trigonus were screened for potential anti-HIV activities in a series of in vitro enzymatic assays, including reverse transcriptase, HIV-1 protease and glycohydrolases. Two extracts of Sutherlandia leaves (SFL-1 and SFL-2) were prepared that inhibited HIV reverse transcriptase and a Lobostemon leaf extract (LTL) was shown to also inhibit this enzyme. All extracts were assayed at 1.25mg/ml. Tannin content was determined for all active extracts using a tannic acid assay. SFL-1 and SFL-2 were found to contain about 6 percent and 7 percent tannins, respectively, and LTL contained 31% tannins by weight. Tannins were removed using polyamide columns and three fractions were collected for each. The extracts were also fractionated with Sephadex G-25, Amberlite IR 120 and Dowex 1-X8 as size exclusion, cation exchange and anion exchange, respectively. Extracts were also fractionated by preparative thin layer chromatography where two compounds were separated from S. frutescens extract with high activity against reverse transcriptase while showing insignificant inhibition towards other enzymes tested. SFL-BFW-10 and SFL-WEF-7 inhibited reverse transcriptase by almost 100 percent and the IC50 values calculated for these compounds were 0.34 and 0.23mg/ml, respectively. Cytotoxicity of these compounds was evaluated on Chang liver cells and peripheral blood mononuclear cells (PBMCs). None of these compounds showed any significant inhibition of cell proliferation. The purity of these compounds could not be confirmed because there was insufficient material to use in the techniques required to show purity and identification. Therefore, TLC was used to determine the nature of these compounds. SFL-BFW-10 was identified as an organic acid and SFL-WEF-7 was identified as flavonoid.

Human immunodeficiency virus (HIV) and its gradual weakening of the immune system is an ever growing threat. Acquired immune deficiency syndrome (AIDS), the final stage of HIV, renders a person vulnerable to various opportunistic infections, which in the end lead to death. Apart from intensive vaccine studies, treatment research mainly focuses on preventing the individual HIV enzymes (reverse transcriptase, integrase and protease) from performing their functions. Entry inhibitors, however, block viral entry into the cell, while antisense drugs lock onto the viral genome to keep it from functioning. In this study production of active recombinant HIV-1 protease and integrase was attempted for future drug screening programs. HIV-1 protease was cloned into a pET28b(+) vector and expressed in ROSETTA(DE3)pLysS cells. The protein was purified using a nickel-affinity column utilizing the hexa-histidine tag encoded by the vector. Gel filtration chromatography was attempted after refolding of the protease, but protease yield seemed to decrease with the additional purification step. Partially purified protease was characterized with kinetic studies. Kinetic parameters of HIV-1 protease were determined to be Km = 592 μM, Vmax = 0.59 μM/min and kcat = 31 s-1. HIV-1 integrase, which was cloned into a pET15b vector, was expressed in E. coli BL21(DE3) cells. The coding sequence had been mutated to introduce the amino acid substitutions F185K and C280S, increasing solubility of the protein. The first step in purification of this protein was nickel-affinity chromatography, after which cation exchange chromatography was attempted. HIV-1 integrase concentration was low throughout experiments and no clear elution from the cation exchange column could be observed. A non-radioactive enzyme linked HIV-1 integrase assay failed to detect integrase activity. Modifications to future studies of the integrase are suggested in the chapter involved.

Thesis (M.CUR.) --University of Limpopo, 2014. / Background: The national HAART programme in South Africa was launched in April 2004. Highly Active Antiretroviral Therapy (HAART) is the medication that slows down the progression from HIV to AIDS, while it had been introduced in Western countries in 1996. Adherence to ART is the major factor in ensuring the virologic success of an initial regimen and is a significant determinant of survival for HIV-infected patients with the wild type virus who are on highly active antiretroviral treatment. Patients with suboptimal adherence are at risk, not only of HIV progression but also of the development of drug resistance and consequent narrowing of options for future treatment. Sub-Saharan Africa carries the highest burden of HIV infections and HIV / AIDS related mortality in the world. South Africa is reported to have the largest population living with the HIV infection. Aims: The aim of the study was to explore factors that contributed to non- adherence of patients to HAART at the Kanyamazane Clinic, Ehlanzeni District, Mpumalanga Province. Study method: A qualitative, exploratory, descriptive, and contextual research design was used for this study. A non-probability purposive sampling method was used to select participants ranging from 15 to 60 years of age and who were on HAART for more than one year. Fifteen participants were selected and the sample size was determined by data saturation. Semi-structured interviews were conducted to collect data through the use of an interview guide on their structured follow-up dates and audio recordings of the interviews were made. Data was analysed following the Tesch’s method. Themes and sub-themes were developed. Results: Findings indicate that factors contributing to non-adherence of patients to HAART are the patient-provider relationship and delivery of services, waiting hours and overcrowding, working hours of the facility, forgetfulness and experiencing better health, belief systems, side-effects, pill burden, migration due to employment, poverty and unemployment, as well as disclosure, stigma, and discrimination. Conclusion and recommendations: The study recommends that HAART services should be provided every day, including on weekends, to improve access and to reduce waiting times; and economic empowerment through skills acquisition programmes to participants and provision of jobs to earn a living. Keywords: Non-adherence, highly active antiretroviral treatment, regimen, drug resistance.

Thesis (M.A. (Clinical Psychology)) --University of Limpopo, 2013 / Studies have indicated that help-seeking behaviour of people living with HIV is not predictable and linear and may entail the utilization of western medicine, traditional medicine and/or complementary medicine. The aim of this study was to explore pre- and post- HIV diagnosis help-seeking behaviour by patients receiving antiretroviral treatment at Witbank Hospital in Mpumalanga Province (South Africa).A qualitative, descriptive phenomenological approach was utilized in the study. Ten participants (male = 5; female = 5, and aged between 30 and 50 years)diagnosed with HIV and who came to the hospital to collect their treatment and for medical review were interviewed using semi-structured interviews. Interpretive analysis method was used to analyse the data. The results suggest the preference for western medicine pre-and post-HIV diagnosis. The results further suggest that help-seeking behaviour is a dynamic process embedded mainly in the conceptualization of the health problem, perception of its severity, the treatment given, and social support experienced.

The worldwide AIDS epidemic is known to have had a profoundly negative social, economic and personal impact and has taken a heavy toll on existing health care systems, particularly in developing countries. South Africa is experiencing an HIV epidemic with enormous social and economic consequences. Lopinavir/ritonavir antiretroviral treatment has been accredited with having a significantly positive effect and is a key advance in controlling HIV morbidity and mortality. An indigenous South African medicinal plant, Sutherlandia frutescens, known for its anti-diabetic properties and immune-boosting effects, is used for treating HIV positive patients suffering from opportunistic infections. Despite the use of the medicinal plant extract as homeotherapeutic medication, there is little evidence of toxicity testing that identifies its potential for interaction with antiretroviral drugs. However, scientific data relating to the mechanism through which Sutherlandia frutescens acts on the immune system has not been comprehensively documented. The aim of this study was to investigate lopinavir/ritonavir induced metabolic abnormalities in rats and whether the introduction of a plant extract of Sutherlandia frutescens would counteract the side effects of ARV medication. The results indicated that the rodents did not become insulin resistant, however, biochemical analysis indicated that extended ARV drug treatment would have caused insulin resistance. Significant morphological changes were found in the livers, kidneys and pancreases of rats exposed to the lopinavir/ritonavir. Rats exposed to the Sutherlandia frutescens plant extract showed improved histopathology with minimal abnormalities.