In response to the urgent call for investigations into antiretroviral (ARV)-African traditional medicine (ATM) interactions, this research was undertaken to ascertain whether chronic administration of the ATM, Sutherlandia frutescens (SF) may alter the bioavailability of the protease inhibitor (PI), atazanavir (ATV), which may impact on the safety or efficacy of the ARV. Prior to investigating a potential interaction between ATV and SF in vitro and in vivo, a high performance liquid chromatography method with ultraviolet detection (HPLC-UV) was developed and validated for the bioanalysis of ATV in human plasma and liver microsomes. An improved and efficient analytical method with minimal use of solvents and short run time was achieved in comparison to methods published in the literature. In addition, the method was selective, linear, accurate and precise for quantitative analysis of ATV in these studies. Molecular docking studies were conducted to compare the binding modes and affinities of ATV and two major SF constituents, Sutherlandioside B and Sutherlandin C, with the efflux transporter, P-glycoprotein (P-gp) and the CYP450 isoenzyme, CYP3A4 to determine the potential for these phytochemicals to competitively inhibit the binding of ATV to these two proteins, which are mediators of absorption and metabolism. These studies revealed that modulation of P-gp transport of ATV by Sutherlandioside B and Sutherlandin C was not likely to occur via competitive inhibition. The results further indicated that weak competitive inhibition of CYP3A4 may possibly occur in the presence of either of these two SF constituents. The Caco-2 cell line was used as an in vitro model of human intestinal absorption. Accumulation studies in these cells were conducted to ascertain whether extracts and constituents of SF have the ability to alter the absorption of ATV. The results showed that the aqueous extract of SF significantly reduced ATV accumulation, suggesting decreased ATV absorption, whilst a triterpenoid glycoside fraction isolated from SF exhibited an opposing effect. Analogous responses were elicited by the aqueous extract and a triterpenoid glycoside fraction in similar accumulation studies in P-gp overexpressing Madin–Darby Canine Kidney Strain II cells (MDCKII-MDR1), which signified that the effects of this extract and component on ATV transport in the Caco-2 cells were P-gp-mediated. The quantitative analysis of ATV in human liver microsomes after co-incubation with extracts and components of SF was conducted to determine the effects of SF on the metabolism of ATV. The aqueous and methanolic extracts of SF inhibited ATV metabolism, whilst the triterpenoid glycoside fraction had a converse effect. Analogous effects by the extracts were demonstrated in experiments conducted in CYP3A4-transfected microsomes, suggesting that the inhibition of ATV metabolism in the liver microsomes by these SF extracts was CYP3A4-mediated. A combination of Sutherlandiosides C and D also inhibited CYP3A4-mediated ATV metabolism, which was in contrast to the response elicited by the triterpenoid fraction in the liver microsomes, where other unidentified compounds, shown to be present therein, may have contributed to the activation of ATV metabolism. The in vitro studies revealed the potential for SF to alter the bioavailability of ATV, therefore a clinical study in which the effect of a multiple dose regimen of SF on the pharmacokinetics (PK) of a single dose of ATV was conducted in healthy male volunteers. The statistical analysis showed that the 90 % confidence intervals around the geometric mean ratios (ATV + SF/ATV alone) for both Cmax and AUC0-24 hours, fell well below the lower limit of the "no-effect" boundary of 0.8 – 1.25, implying that the bioavailability of ATV was significantly reduced in this cohort of subjects. It may thus be concluded that if the reduction in bioavailability observed in this clinical study is found to be clinically relevant, co-administration of SF commercial dosage forms and ATV in HIV/AIDS patients may potentially result in subtherapeutic ATV levels, which may in turn contribute to ATV resistance and/or treatment failure. This research has therefore highlighted the potential risk for toxicity or lack of efficacy of ARV regimens which may result when ATMs and PIs are used concurrently and that patients and health care practitioners alike should be aware of these perils.

Magister Pharmaceuticae - MPharm / Many studies have reported low cure rates for multidrug-resistant tuberculosis (MDRTB) patients and MDR-TB patients co-infected with human immunodeficiency virus (HIV). However, little is known about the effect of HIV infection and antiretroviral therapy on the treatment outcomes of MDR-TB in South Africa. Therefore, the objectives of the study are: to find out whether HIV infection and interactions between ARVs and second line anti-TB drugs have an impact on the following MDR-TB treatment outcomes: cure rate and treatment failure at Brewelskloof Hospital. MDR-TB patients were treated for 18-24 months. The study was designed as a case-control retrospective study comparing MDR-TB treatment outcomes between HIV positive (cases) and HIV negative patients (controls). Patients were included in the study only if they complied with the following criteria: sensitivity to second line anti-TB drugs, MDR-TB infection, co-infection with HIV (for some of them), male and female patients, completion of treatment between 1 January 2006 and 31 December 2008. Any patients that presented with extreme drug-resistant tuberculosis (XDR-TB) were excluded from the study. Data were retrospectively collected from each patient’s medical records. There were a total of 336 patients of which 242 (72%) were MDR-TB patients and 94 (27.9%) MDRTB co-infected with HIV patients. Out of the 242 MDR-TB patients, 167 (69.2%) were males and 75 (30.7%) were females. Of the 94 patients with MDR-TB co-infected with HIV, 51 (54.2%) males and 43 (45.7%) females. Patients with multidrug-resistant tuberculosis co-infected with HIV who qualify for antiretroviral therapy were treated with stavudine, lamivudine and efavirenz while all MDR-TB patients were given kanamycin, ethionamide, ofloxacin, cycloserine and pyrazinamide. The cure rate of MDR-TB in HIV (+) patients and in HIV (-) patients is 34.5% and 30 % respectively. There is no significant difference between both artes (pvalue = 0.80). The MDR-TB cure rate in HIV (+) patients taking antiretroviral drugs and in HIV (+) patients without antiretroviral therapy is 35% and 33% respectively. The difference between both rates is not statistically significant. The study shows that 65 (28.0%) patients completed MDR-TB treatment but could not be classified as cured or failure, 29 (12.5%) patients failed, 76 (32.7%) defaulted, 18 (7.7%) were transferred out and 44 (18.9%) died. As far as treatment completed and defaulted is concerned, there is no significant statistical difference between HIV (+) and HIV (-) The number of patients who failed the MDR-TB treatment and who were transferred out is significantly higher in the HIV (-) group than in the HIV (+) group. Finally the number of MDR-TB patients who died is significantly higher in the HIV (+) group). The median (range) duration of antiretroviral therapy before starting anti-tuberculosis drugs is 10.5 (1-60) months. According to this study results, the MDR-TB treatment cure rate at Brewelkloof hospital is similar to the cure rate at the national level. The study also hows that HIV infection and antiretroviral drugs do not influence any influence on MDR-TB treatment outcomes. / South Africa

The research undertaken in these studies aimed to investigate the feasibility of developing and manufacturing innovative solid lipid carriers, such as solid lipid nanoparticles (SLN) and/or nanostructured lipid carriers (NLC) using a hot high pressure homogenization method, for didanosine(DDI). In addition, studies using in vitro differential protein adsorption were undertaken to establish whether the SLN and/or NLC have the potential to deliver DDI to the central nervous system (CNS). Prior to initiating pre-formulation, formulation development and optimization studies of DDI-Ioaded SLN and/or NLC, it was necessary to develop and validate an analytical method for the in vitro quantitation and analysis of DDI. An accurate, precise and sensitive RP-HPLC method with UV detection set at 248 nm was developed, optimized and validated for the quantitative in vitro analysis of DDI in formulations. Pre-formulation studies were designed to evaluate the thermal stability of DDI and to select and characterize lipid excipients that may be used for the manufacture of the nanocarriers. It was established that DDI is thermostable at temperatures not exceeding 163°C and therefore a hot high pressure homogenization technique could be used to manufacture DDI-loaded SLN and/or NLC. Lipid screening studies revealed that DDI is poorly soluble in both solid and liquid lipids. A combination of Precirol® ATO 5 and Transcutol® HP was found to have the best solubilizing-potential for DDI of all lipids investigated. The inclusion of Transcutol® HP into Precirol® ATO 5 changed the polymorphic form of the solid lipid from the stable 13-modification to a material that exhibited the co-existence between α- and β-polymorphic forms. The relatively high solubility of DDI in Transcutol® HP compared to Precirol® ATO 5 was an indication that a solid lipid matrix prepared from a binary mixture of Precirol® ATO 5 and Transcutol® HP was likely to have a higher loading capacity and encapsulation efficiency for DDI than a matrix consisting of Precirol® ATO 5 alone. Furthermore, the potential for the solid lipid matrix to exist in the α- and/or β-modifications when Transcutol® HP was added to Precirol® ATO 5 suggested that expulsion of DDI from a solid lipid matrix during prolonged storage periods was likely to be minimal. Therefore it was considered logical to investigate the feasibility of incorporating DDI into NLC and not in SLN. However, due to the limited solubility of DDI in lipids, formulation development of DDI-loaded NLC commenced using small quantities of DDI. Formulation development and optimization studies of DDI-loaded NLC were initially aimed at selecting a surfactant system that was capable of stabilizing NLC in an aqueous environment. Solutol® HS alone or a ternary mixture consisting of Solutol® HS, Tween® 80 and Lutrol® F68 was found to stabilize the nanoparticles in terms of particle size and the polydispersity index. The use of the ternary mixture as the surfactant system was preferred to using Solutol® HS alone as Lutrol® F68 and especially Tween® 80 have been successfully used to target the delivery of API to the brain. Aqueous DDI-free and DDI-Ioaded NLC containing increasing amounts of DDI were manufactured using hot high pressure homogenization at 800 bar for three cycles. The NLC formulations were characterized in terms of particle size, polydispersity index, zeta potential, and polymorphism, degree of crystallinity, encapsulation efficiency (EE), shape and surface morphology. The mean particle size for all formulations was below 250 nm with narrow polydispersity indices, indicating that narrow particle size distribution had been achieved. The d99% values for all formulations tested, were generated using laser diffractometry, and were below 400 nm, with span values ranging from 0.84 - 1.19 also suggesting that a narrow particle size distribution had been achieved. The zeta potential values measured in double distilled water with the conductivity adjusted to 50 μS/cm ranged from -18.4 to -11.4 mV. In addition, all the formulations showed a decrease in the degree of crystallinity as compared to the bulk lipid material and WAXS shows that the formulations existed in a single β-modification form. Furthermore DDI that had been incorporated into the NLC appeared to be molecularly dispersed in the lipid matrices. These parameters remained unaffected for most formulations following storage for two months at 25°C. In addition these formulations contained a mixture of spherical and non-spherical particles irrespective of the amount of DDI that was added during the manufacture of the formulations. These studies showed that it was feasible to develop and incorporate small amounts of DDI into NLC. However in order to use these delivery systems for oral administration of DDI to paediatric patients, strategies to improve the amount of DDI that could be loaded into the particles and to achieve high encapsulation efficiencies had to be developed. The limited solubility of DDI in lipid media was identified as a major factor that affected the loading capacity and encapsulation efficiency of DDI in the NLC. Therefore, a novel strategy aimed at increasing the saturation solubility of DDI in the lipid by attempting to increase the dissolution velocity of the drug in the lipid using a particle size reduction approach, was designed and investigated. DDI was dispersed in Transcutol® HP and the particle size of DDI in the liquid lipid medium was reduced gradually using hot high pressure homogenization and the product obtained from these studies was used to manufacture DDI-loaded NLC using a cold high pressure homogenization procedure. Although the encapsulation efficiency and drug loading following use of this approach was relatively high, the particles were large and showed a tendency to grow in size leading to the formation of microparticles after storage for two months at 25°C. In addition, the degree of crystallinity of the nanoparticles increased rapidly over the same storage period which led to expulsion of DDI nanoparticles for the NLC, despite the DDI loading in NLC being unaffected. It was clearly evident that this new approach of manufacturing solid lipid nanocarriers could be used as a platform not only for enhancing the loading capacity of DDI in solid lipid nanocarriers but also for other hydrophilic drugs. Differential protein adsorption patterns of DDI-loaded NLC were generated in vitro using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in order to establish the potential for these systems to deliver DDI to the CNS. NLC formulations containing small amounts of DDI were used as these formulations showed a better stability profile than the formulation with a higher encapsulation efficiency and drug loading capacity. Furthermore, the encapsulation efficiency and drug loading of DDI were considered sufficient for use in 2-D PAGE studies. Data obtained from 2-D PAGE analysis reveal that DDI-loaded NLC preferentially adsorb proteins in vitro that are responsible for specific brain targeting in vivo. More importantly, these studies reveal that in addition to Tween® 80 that has already been shown to have the potential to target CDDS to the brain, Solutol® HS 15 has the potential to achieve a similar objective. Consequently, DDI-loaded NLC have the potential to deliver DDI to the brain and these results may be used as a platform for conducting in vivo studies to establish whether DDI can cross the blood brain barrier and enter the CNS when administered in NLC which may in turn lead to a major breakthrough in the management of HIV/AIDS and Aids Dementia Complex (ADC).

Antiretroviral drugs taken during pregnancy, reduce the rates of mother-to-child transmission from 35 percent to as low as 1 to 2 percent (UNAIDS, 2009). In 2002, the Prevention of Mother-to-Child Transmission (PMTCT) programme was implemented in South Africa. Studies on the implementation of the PMTCT programme have shown that understaffed and under-developed health care facilities were key barriers to the provision of PMTCT services (Health Systems Trust, 2002: 6; Skinner et al., 2003). The aim of this study was to assess the challenges experienced by health care workers working in public sector facilities in the Nelson Mandela Metropole after implementation of the dual therapy PMTCT programme. Four areas were investigated: Infrastructure; Drug Supply Management; Clinic Procedures and Staffing. A quantitative descriptive study was conducted in August 2009 at nine public health care facilities in the Nelson Mandela Metropole, South Africa. Questionnaires were issued to 81 nurses and 41 pharmacy personnel (pharmacists and pharmacist assistants). Checklist audit forms were issued to the Facility Manager of each facility and completed with the researcher. The key findings for Infrastructure were lack of space at patient waiting rooms (9; 100 percent n=9), counselling area (5; 55.5 percent; n=9), nurse consultation rooms (6; 66.6 percent; n=9), storage areas (5; 55.5 percent; n=9) and filing areas (7; 77.7 percent; n=9). The key findings for Drug Supply Management were none of the dispensaries (0 percent; n=10) were fully compliant with Good Pharmacy Practice, pharmacy personnel indicated that there were no stock cards for medication (13; 31.7 percent; n=41); there was less than two weeks supply of buffer stock kept for zidovudine and nevirapine (13; 35.1percent; n=37) and medication orders were placed without any reference to minimum and maximum levels of medication (15; 36.5 percent; n=41) . The key findings for Clinic Procedures were only two facilities followed up on patients that had missed appointments (22.2 percent; n=9) and four facilities (44.4 percent; n=9) had a tracking system for patients that had defaulted. Of the nine facilities only three (33.3 percent; n=9) updated patient demographic details regularly. The key findings for Staffing were a shortage of doctors, nurses, counsellors and pharmacists at the facilities. One of the major challenges identified was the lack of training offered on new PMTCT protocols with 56.2 percent (45; n=80) of the nurses stating that no training was provided on the dual PMTCT protocol. Only 54.3 percent (44; n=81) of nurses stated that they knew the criteria to start the mother on dual PMTCT therapy. In conclusion there is an urgent need for barriers such as lack of staff, lack of space, lack of training on PMTCT and standard procedures for follow up of patients to be addressed in order to ensure the successful scaling up of PMTCT.

The Human Immunodeficiency Virus (HIV)/Acquired Immune Deficiency Syndrome (AIDS) is one of the leading chronic diseases affecting people in South Africa and throughout the world. This study aimed to investigate the effect disclosure of HIV status had on antiretroviral therapy (ART) adherence. A convenience sample of 65 HIV positive adult patients currently taking ART at a public Primary Health Care (PHC) clinic in the Nelson Mandela Metropole was selected. Participation was voluntary and confidentiality was maintained at all times. Data was collected using three tools/techniques: (1) a Patient Questionnaire (PQ) to extract information on patient's demographics, HIV disclosure status, regimen the patient was on and self-reported adherence to ART; (2) an audit of a Patient Medical Record (PMR) for information on the regimen the patient was on, the period during which the patient had been on ART medication, the adherence to ART care and the level of the patient‟s biological markers; and (3) Pill Counts (PC) performed on the patient's medical supply to validate the self-reported adherence to ART. There was no significant relationship between the disclosure of HIV status and adherence to ART (p= 0.59; Chi²). However, the relationship between the adherence to ART and increase in the CD4 count levels of patients on ART in this population was significant (p=0.03; Chi²). It can be concluded that no direct relationship was found between the disclosure of HIV status and adherence to ART in this population. However, several factors affected the reasons and decisions of individuals to disclose their HIV status and this influenced their daily taking of medication.

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.

Thesis (MPH.) -- University of Limpopo, 2019 / Virological failure occurs when the viral load fails to supress to undetectable limit and immunological failure is when the immune system fails to improve with the CD4 count remaining low on clients on antiretroviral drugs. These being markers of poor adherence to antiretroviral drugs or treatment failure. Upon routine blood-monitoring of students on antiretroviral drugs, the researcher noticed that some students’ viralload levels were not suppressing and their immune system was not improving. The purpose of the study was to identify the experiences of those students whose viral load is not suppressing and their immune system not improving. The objective was to identify and describe the experiences of students with immunological and virological failure on antiretroviral drugs at the University of Limpopo. A qualitative, explorative and descriptive study design was used. Convenience purposive sampling method was adopted. Using a semi-structured interview guide, face-toface interviews were conducted on 10 students on antiretroviral drugs at the Student Health and Wellness Centre, University of Limpopo. Techs’ method was used to analyse data. Guba’s model for establishing trustworthiness was used. The study yielded the following themes: Disclosure, stigma, antiretroviral drugs packaging, side effects of antiretroviral drugs and service delivery. The study concluded that students are afraid to take their treatment for fear of stigmatisation and disclosure is still a problem. Students tend to forfeit taking drugs when studying for examinations due to side effects of the drugs. It is recommended that service delivery and antiretroviral drugs packaging be user friendly. Key words: Disclosure, stigma, side effects, antiretroviral drugs packaging and antiretroviral drugs.

Background: HIV transmission is greatly reduced when antiretroviral treatment (ART) suppresses an infected person’s HIV viral load. It is unclear, however, whether the contextual risk of incident HIV is optimally reduced by widespread individual-level suppression of HIV viral load alone or in combination with other HIV prevention services. HIV service coverage and community norms can influence risk in small area geographies; and contextual factors, like gender inequality and stigma, may foster environments conducive to HIV transmission. Yet, the relationship between places with high HIV levels and the clustering of area risk factors is unknown. The goal of this dissertation is to learn if and how a geographically focused combination implementation strategy could reduce population-level HIV risk. Analyses explored whether small area risk profiles explain area differences in HIV. The guiding hypothesis is that in high HIV prevalence settings, low HIV service uptake in a geographically defined area increases the prevalence of high HIV viremia, leading to greater HIV transmission and incident HIV. Methods: A systematic review was conducted to examine the association between population-level measures of HIV viral load and incident HIV infection in generalized and concentrated epidemics. Publications were English, peer-reviewed articles published from January 1, 1995 through February 15, 2019 that explicitly defined HIV viral load and assessed outcomes of HIV recency, incidence, seroconversion, or new diagnosis. Studies sampled general or key populations through population-based surveillance registries, household-based enumeration, cluster sampling, or respondent driven sampling. Descriptive statistics summarized review findings. The Swaziland HIV Incidence Measurement Survey (SHIMS) data were used for the remaining analyses. Using a two-stage cluster-based design, a nationally representative, household-based sample of adults, ages 18-49 years was enrolled from December 2010 to June 2011 in Eswatini. Consenting adults completed an interview and received home-based rapid HIV testing and counseling. All seropositive samples were tested for HIV viral load using the COBAS AmpliPrep/Taqman HIV-1 Test, v 2.0. Adults testing HIV-seronegative were enrolled in a prospective cohort for the direct observation of HIV seroconversion, completing an interview and home-based rapid HIV testing six months later. Multi-level latent class modeling was performed to identify statistically significant combinations of HIV risk factors and to classify the combinations into small area risk profiles. In the cross-sectional sample, linear regression with robust standard errors assessed the correlation between area profiles and places with high levels of uncontrolled HIV infection, or HIV core areas, measured by the area prevalence of detectable virus (≥20 copies/milliliter) among HIV-positive adults and among all adults, regardless of HIV status. In the prospective cohort, generalized linear regression of longitudinal data assessed the association between area profiles and places prone to new HIV infections (i.e., HIV susceptible areas), measured by area-level HIV seroconversions. Results: The systematic review found an evidence base primarily of lower quality studies and inconsistent HIV viral exposure measurement. Overall findings supported a relationship between increasing levels of suppressed HIV in HIV-infected populations and fewer new infections over time. Better quality studies consistently showed higher population viremia (i.e. HIV viral quantity among all persons, regardless of HIV status) associated with HIV incidence in high prevalence populations; population viral load (i.e., HIV viral quantity among only HIV-positive persons) did not show an association with incident HIV in high prevalence, general populations and was inconsistent in key populations. To determine whether area risk profiles can pinpoint HIV core areas, latent class modeling was used to categorize 18,172 adults into one of six HIV risk types. The risk typology, classified through unique combinations of HIV service uptake and sexual risk behaviors, conveyed an adult’s propensity for HIV transmission and/or acquisition risk. The model next identified the area-level composite prevalences of HIV risk types; estimated the three most frequent, unique composite combinations; and categorized them into area risk profiles characterizing HIV risk: low-moderate acquisition risk, moderate acquisition/transmission risk, and high acquisition/transmission risk. The high acquisition/transmission areas comprised the largest proportions of highest risk transmission and acquisition types. The prevalence of detectable viremia progressively increased from low-moderate acquisition, moderate acquisition/transmission, and high acquisition/transmission profiles [17.7%, 25.4%, and 35.1%, respectively]. When compared with low-moderate acquisition areas, the prevalence of detectable viremia was 7.4% [p<.001] higher in moderate acquisition/transmission areas and 17.1% [p<.001] higher in high acquisition/transmission areas. The prevalence of detectable viral load significantly decreased from low-moderate acquisition to moderate acquisition/transmission areas [76.6% versus 68.7%, p<.001], and was significantly higher in high acquisition/transmission areas by 7.3% [p<.001], when compared with low-moderate acquisition areas. To determine whether area risk profiles can predict HIV susceptible areas, a total of 18,172 adults were surveyed of which 4396 [24%] had detectable viremia. 11,880 [96%; n=12,357] HIV-seronegative adults enrolled in the prospective cohort and 11,155 [94%] of them completed an endline visit. Four area profiles were identified, defined by unique patterns in prevalence of HIV viremia and of sexual risk behaviors. The proportion of HIV susceptible areas progressively increased from Profiles A, B, C, and D [14.3%, 21.8%, 24.6%, and 30.8%, respectively]. HIV susceptible areas were more than twice as likely to occur in Profile D than Profile A environments [RR 2.13, 95% confidence interval (CI) (1.13, 4.00); p=0.02]. Profile D areas had prevalences of unknown partner HIV status and detectable viremia at 28% and 24%, respectively. In contrast, Profile A areas had prevalences of only 8% with unknown HIV status and 31% with detectable viremia. Conclusion: This dissertation shows that geographic risk profiles can explain differences in population-level HIV outcomes. Risk factors spatially cluster in predictable, meaningful combinations that can inform an area typology of HIV risk. The co-location of adults predisposed to greater HIV risk may heighten levels of uncontrolled HIV infection, thereby creating potential area sources of ongoing transmission; however, the concurrent levels of other risk factors may have more influence in reducing population-level incidence than previously considered. A composite indicator of contextual HIV risk may reveal places core to HIV transmission and susceptible to HIV acquisition. Such area profiles may help identify the combination of locally specific risk factors that readily promulgate HIV and better inform the design of place-based HIV intervention packages to enhance current strategies towards global HIV control.

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