A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2016 / Introduction South Africa has the largest absolute number of individuals living with human immuno-deficiency virus (HIV) in the world. The quality assurance (QA) of HIV rapid diagnostic tests (RDT) has not kept pace with the rate of expanded testing and utilisation of RDT. This has made it difficult to assess the accuracy of testing. In South Africa HIV counselling and testing (HCT) and the use of HIV RDT is the point of entry to HIV prevention, management, care, treatment and support. HCT in public health facilities is delivered mainly through rapid testing by nonprofessional staff. Implementation of QA processes is crucial for accurate diagnosis of HIV. However, accuracy of HCT using rapid test kits in non-laboratory settings in South Africa will remain a challenge unless there is evidence that nonlaboratory rapid HIV testing results are as reliable as the laboratory-based enzyme immunoassays. This study aimed to determine the accuracy of HIV RDT in the context of an intervention. The objectives of the study were: i. To assess the sensitivity and specificity of rapid test kits in two provinces; ii. To assess the sensitivity and specificity of rapid test kits between the two provinces and New Start nongovernmental organisation (NGO) which implemented a more comprehensive quality management system (QMS); iii. To assess the accuracy of HIV RDT in the two provinces; iv. To assess the accuracy of HIV RDT between the two provinces and New Start sites. The hypothesis was ‘the accuracy of HIV diagnosis using HIV RDT kits in nonlaboratory settings in which an intervention has been introduced (internal quality control), also known as IQC, will not be different compared to settings that do not utilize IQC’. Methods In South Africa, the current laboratory-based gold standard for diagnosis of HIV infection in adults in the public sector as recommended by the National Health Laboratory Services (NHLS) Virology expert committee is a serial 2-test algorithm. Thus, a reactive enzyme immunoassay (EIA) test result must be confirmed by a second confirmatory EIA that must be different in terms of antigens and technology. The Expert Committee recommendation is that positive results should be confirmed by a separate sample 14 days later. In the case of HIV rapid testing the national HIV counselling and testing (HCT) policy, 2010, similarly recommends a serial 2-test algorithm for diagnosis where a reactive screening test is confirmed by a different confirmatory test. If the confirmatory test is reactive the diagnosis is positive. If test 1 is non-reactive then the diagnosis is negative. In case of discrepant results an enzyme-linked immunosorbent assay (ELISA) test was recommended as a tiebreaker. A new HIV testing services (HTS) policy was approved in South Africa in 2016 and it further recommended that the first time discrepant results are found, the counsellor must repeat the algorithm and if on repeat, the results are still discrepant, then reflex testing is recommended where the blood (whole blood) of a client is taken to the laboratory for ELISA (NDOH, 2016).This algorithm has replaced the use of Western Blot is South Africa. The rationale for the change was based on the sensitivity and specificity of 3rd and 4th generation ELISAs, workload, costs and expertise. With the introduction of the 3rd and latterly 4th generation EIA tests the above algorithm is in use in South Africa and has replaced the use of the Western blot as a confirmatory test. The rationale for the change is based on earlier detection of HIV infection, workload, costs and expertise. Further developments for a diagnostic algorithm include the use of a fourth generation test and if reactive to use a HIV-1 and HIV-2 discriminatory test and HIV viral load. This study was cross-sectional and compared the performance of HIV RDT in selected sites in Limpopo province that had introduced an intervention viz., an internal quality control (IQC) as part of quality management system (QMS) implementation, and compared to Mpumalanga province that had not introduced the IQC and performed limited QMS activities. The sample size calculated for the study was N = 717. IQC is an independent internal quality control that is used to check that an analytical phase or test precision is optimal. The introduction of routine QMS in Limpopo was through implementation of IQC supported by appropriate training and certification of implementers. IQC was implemented routinely as part of the provincial QA initiatives with the aim of supporting the implementation of HIV RDT in non- laboratory settings. There are other QA measures that may be implemented to support HIV RDT programmes including external quality assessment (EQA) such as proficiency testing (PT) which is a tool used to assess the testing process independently. EQA implementation was however not part of the Limpopo (LP) QMS implementation. Six high volume testing sites comprising of 3 hospitals and 3 clinics were selected per province. This was to avoid the risk of not meeting the required number of participants due to refusals, lack of results and challenges with reporting. In order to mitigate risk, the study was oversampled, where a total of 457 participants from the LP sites were enrolled in the study and results were analysed and compared to those of 361 participants from the Mpumalanga (MP) sites resulting in a total sample size of 818. The analyses included demographics, performance of RT as measured by the number of discordant results, reliability and validity of rapid tests RT as measured by the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) results. The data between Limpopo and Mpumalanga were further analysed together with the data from selected sites from a non-governmental organisation (NGO) called New Start and the performance, reliability and validity of the HIV test results were compared. The main role of New Start was to offer HCT in support of the government priorities and it implemented several different QMS measures for HIV rapid testing, namely, IQC, EQA, PT and re-testing, training for implementers, development and implementation of standard operating procedures (SOPs), and ensuring that all commodities were stored under appropriate conditions including temperature monitoring. In order to determine the validity and reliability of HIV RDT against the gold standard ELISA in Limpopo, Mpumalanga and New Start sites, the rate of discordance, the sensitivity, specificity, PPV and NPV were determined. Logistic regression models were constructed to assess the association between the interventions in the provinces. Crude and adjusted odds ratios were used as a measure of association between exposure and outcome and a 95% precision of estimate was used to ascertain statistical significance. Exposure factors with p<0.05 were considered statistically significant. Results A total of 947 attendees for HCT services in selected sites in Mpumalanga and Limpopo provinces between August and April 2012, were screened and of these, 818 were enrolled into the study according to the study inclusion criteria. There was no significant difference (p=0.05) between the number of participants enrolled in Limpopo (457) as compared to Mpumalanga (361) though Limpopo enrolled more participants than Mpumalanga. All available data from New Start sites for the period 2008 was analysed. The gender, rate of discordance and HIV positivity rate were significantly different between the two provinces (p<0.05). The study showed that the laboratory-based HIV prevalence rate in each setting was 22.9% in Limpopo, 26% in Mpumalanga and 11% in New Start sites. The prevalence rates reported by Shisana, 2014, were 21.8% for Mpumalanga and 13.9% for Limpopo. The rate of discordant HIV test results between the 2 provinces and New Start sites was also measured where discordant results were defined as those that were different between HIV rapid test and the ELISA test. The rate of discordant HIV test results was 5.9% (27) in Limpopo, 11.0% (40) in Mpumalanga p= 0.010 and 1.4% (68) in New Start sites. False negative results accounted for all the discordant results. Logistic regression models were used to estimate the Odds Ratio (OR) and the 95% confidence interval of the association between implementation of QA programme and the HIV test accuracy or the HIV discordance rate. Facilities without a QA intervention programme had an approximately 2-fold increased odds of HIV test discordance compared to facilities with a QA programme in place (crude OR 1.86, 95% CI: 1.10 – 3.12 and adjusted OR 1.90, 95% CI:1.08 - 3.30). This association was statistically significant. The sex and age of the participants was not associated with discordance rate. The sensitivities of the HIV RDT in Limpopo, Mpumalanga and New Start sites were 86% (CI: 83.9-89.4), 72% (CI: 64.2-79.0) and 98% (CI: 97.6-98.4) respectively. In this study, specificity ranged within 99% (CI: 98.9-99.9) in all sites (Provinces and New Start sites). The PPV in Limpopo, Mpumalanga and New Start sites were 98% (CI: 93.2-99.6), 97% (CI: 91.0-99.2) and 93% (CI: 92.3-93.7) respectively, The NPV results in Limpopo were 93% (90.5-95.2), Mpumalanga at 86% (CI:81.3-90.7). For New Start sites, the NPV was 99.6% (CI: 99.4-99.8). The sensitivities and specificities of the sites were used at a national prevalence rate of 18.8% to determine the national PPV and NPV and these were found to be 100% (CI: 100-100) and 91.3% (CI: 89.04-92.96) respectively. Discussion In all three settings the World health Organisation (WHO) recommended sensitivity (>99%) and specificity (>98%) were not met. There was a gradient of sensitivities and specificities that was associated with the extent of QA implementation. Thus, New Start sites with a more extensive set of QA activities had the highest sensitivity; LP with introduction of IQC, had an intermediate sensitivity and MP the lowest. Despite the introduction of an intervention LP was not able to meet the required level of QA implementation compared to New Start. Increased discordance was associated with the extent of implementation of QA as shown by the results of the logistic regression model (crude and adjusted). In this study there was a decline in sensitivity that resulted in some false negative results. To a lesser extent, some false positive results were also identified in New Start sites. In the case of LP and MP the potential contributory factors to false negative results xi would include the extent of QA implementation and training. Further evidence of the relative poor implementation would include the M&E assessments and in the course of the study there lost results, poorly taken and missing specimens that led to data being excluded. Conclusion On the basis of these results, it is concluded that implementation of quality assurance measures is critical to ensure correct diagnosis of rapid HIV testing. Furthermore, implementation of a combination of aspects of QA is urgently required including training of all implementing staff on quality assurance of rapid HIV testing, monitoring and evaluation to assess kit performance through IQC and PT, as well as implementation of the current South African HIV testing Services (HTS) Policy. All PT methods should be explored for implementation and training and certification of implementers must be ensured. / MT2017

A research report submitted to the School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, in partial fulfillment of the requirements for the degree of Masters in Science in Epidemiology and Biostatistics June 2017 / Introduction Delays in diagnosing and treating tuberculosis increase the risk of transmission, morbidity and mortality especially in low socio-economic settings with high HIV and TB rates. The aim of this study was to determine factors associated with the delay in the diagnosis and treatment of suspected TB by HIV status in hospitalised patients in South Africa. Methods This study was a secondary analysis of data from a three centre prospective cohort of inpatients recruited between 2006 and 2009 that were clinically diagnosed with active TB on admission. Results Data from 1018 patients (67% female) of a median age of 36 years (IQR: 30-44) with known HIV status were analysed: 875 (86%) positive and 143 (14%) negative. HIV positive patients had significantly longer median total delays relative to the negative (39 days, IQR: 28-74 vs. 32 days, IQR: 21-56; p<0.02). Unemployment, seeking prior treatment and use of cotrimoxazole predicted total delay in the HIV positive patients. Conclusion Patient delay is high in HIV positive patients compared to the HIV negative. Public health interventions targeting earlier diagnosis of TB disease in HIV positive patients should be enhanced. / MT2017

The South African HIV testing guideline, Center for Disease Prevention and Control (CDC), World Health Organisation (WHO), and Joint United Nations Programme on HIV/AIDS (UNSAID) have recommended that routine HIV testing be offered in every healthcare facility. The emergency department(ED) is uniquely placed to be involved in this initiative due the volumes and characteristics of patients seen in the ED. This study seeks to determine the attitude of ED staff and their perceived barriers towards routine testing in the ED. Methods: Paper-based questionnaires were distributed to 170 members of ED staff in 3 academic hospitals. Survey Questionnaires contained 25 questions to reflect staff knowledge of HIV infection, their attitude towards testing, current testing practices and perceived barriers to testing. Chi square test was used to test for associations between various variables and the willingness to test. Result: Response rate was 52% (88/170). Average year of experience in an ED (SD) was 4.4 years. Only 30% of ED staff favoured routine testing in the ED. However, 63% of staff was willing to test if result was available within 20 minutes. Members of ED staff generally prefer that a HIV counsellor disclose the result of a positive test. Members of the white race and those who identified fewer barriers were more likely to test. Important barriers cited include; time constraints (77%), inadequate resources (77%), and lack of support staff (71%). Conclusion: The ED staff generally favoured risk based testing over routine testing. Members of the ED staff are generally willing to offer routine HIV testing, but the presence of barriers may limit the implementation of routine HIV testing in the ED.

MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand, 2009 / The HIV-1 Vpu protein functions largely to target CD4 molecules for proteasomal degradation, and to enhance virion release. The subcellular localisation of Vpu is related to these functions. Previous studies showed subtype B Vpu localisation at the ER/Golgi complex, while subtype C Vpu localised to the plasma membrane (PM) at 48 hours post-transfection. To determine if subtype C Vpu can localize to the ER/Golgi, we evaluated the cellular localisation of Vpu from two South African subtype C isolates as compared to subtype B Vpu, over time. Codon optimized vpu genes from subtype C isolates FV5 and FV15 (which have a six and two amino acid insert in the N-terminal domain, respectively) and a representative subtype B vpu were TA cloned into the pcDNA6.2/C-emGFP expression vector. The three VpuemGFP recombinant plasmids were cotransfected with pDsRed-ER, pDsRed-Golgi, or pDsRed-Mem into HEK 293T cells, and observed at 24, 48, and 60 hours posttransfection under a confocal microscope to confirm the presence of Vpu at different subcellular compartments. Cotransfection and microscopy conditions were methodically optimised. At 24 hours post-transfection, the subtype C FV5 Vpu had ER/Golgi localisation, but none at the PM. The subtype C FV15 Vpu had weaker ER/Golgi localisation and no PM localisation. In contrast, the subtype B Vpu had strong PM localisation. At 48 hours, FV5 and FV15 Vpu showed PM localisation, while subtype B Vpu was clearly localised at the ER/Golgi. At 60 hours, FV5 Vpu was observed at the PM, whereas FV15 and subtype B Vpu showed ER/Golgi localisation. These findings illustrate the efficient translocation of Vpu between different cellular compartments and for the first time, the difference in timing between subtype B and C Vpu, as well as íntrasubtype differences. This difference in shuttling suggests implications for the timing of viral assembly and release. Further investigations may clarify the impact of this timing on the difference in disease pathogenesis noted between infections with the different subtypes.

This report has been submitted to the School of Public Health in the Faculty of Health Sciences of the University of the Witwatersrand. The report is in partial fulfillment of the requirements for the degree of Master of Public Health in the field of Social Behaviour Change and Communication (MPH). Date: 19 June 2017 / Background Extant research in South Africa and elsewhere has shown that men tend to shy away from health care services. For men living with HIV, this may lead to poor ART adherence. Literature on men and masculinities suggests that social definitions of what it means to be a „man‟ affect and influence men‟s decisions to either seek or not seek health care. Some authors have argued that these definitions of manhood often discourage men from using public health facilities. Another body of scholarship suggests that South African men‟s poor health seeking behaviours and adherence to ART is also influenced partly by their experience with the healthcare system and the attitude of healthcare providers. Aim This study explored health seeking behaviours of men living with HIV with a focus on their long-term experiences with HIV care and treatment services, as well as their experiences with the new single, fixed-dose combination tablet and how it influences adherence to ART in South Africa. Methods This study used an exploratory qualitative study design, using in-depth interviews (IDIs) to collect data. The IDIs were conducted with 14 adult HIV positive men living who had been on ART for five or more years and were receiving care and treatment at Stretford Community Health Centre (CHC) in Orange Farm, Johannesburg. Findings The men shared in-depth individual experiences of what it means to live with HIV and subsequently being on treatment for a long time (five or more years). They gave insights on the psychosocial challenges that they have endured living with HIV. Furthermore, they shared their subsequent resolve to live healthy lifestyles despite the challenges they have encountered related to living with HIV. Regarding health care systems, the men were generally impressed with the services received at the health facility. Further, all but one man, were not bothered about the gender of the nurse attending to them at the health facility. The experiences of the men collectively, reflect and provide some insights of how self-determination can transcend societal definitions of what it means to be a man, and how notions of masculinities may influence how men cope and adjust to living with HIV, and their adherence to ART over a long period of time. Conclusions This study concludes that men are capable of modifying their masculine positions to cope with life-long adherence to HIV treatment regimens. The men‟s self-determination to adjust from destructive notions of masculinities in order to accommodate treatment is an area for further research to inform men‟s programming. Social support systems are key to men‟s ART adherence programmes. Stigma and discrimination remains a challenge within communities, and as such community sensitisation programmes should be strengthened to combat the vice. Programmes targeting men should promote behaviours perceived to be beneficial in prolonging men‟s lives. The introduction of the fixed dose combination pill provides a great opportunity to strengthen men‟s abilities to consistently adhere to ART as it was taken once a day. / MT2017

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree Master of Science. Johannesburg 2015 / The controversy surrounding the findings that copy number variation (CNV), of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. In this study, we evaluated the standard method of quantitative Real-Time PCR (qPCR) and Droplet Digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR, with the addition of another 30 black individuals to the ddPCR cohort. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet Digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course. We further used the CCL4L copy number data to examine variation of these genes with reference to measured stimulated CCL4 production by the same individuals. Although significant differences in the copy number range medians and patterns of distribution of the genes CCL4L1, CCL4L2 and combined CCL4L were observed between the two populations, on a whole, the two populations do not differ significantly with respect to CCL4 production. Caucasian females however had a higher level of protein production per copy of CCL4L than Black females. When stratifying production based on population specific copy number median, Black individuals showed a decreased level of protein production at a CCL4L copy number below the median, although this was not maintained when the CCL4L1 and CCL4L2 genes were analysed individually. CCL4 copy number and production data was compared to data generated for CCL3 in the same cohort. CCL4 chemokine levels were significantly higher in both the Black and Caucasian populations and Black individuals had a higher number of gene copies of the CCL3L genes giving rise to functional CCL3 protein than the CCL4L genes producing functional CCL4 protein. These results highlight genetic differences between divergent populations, differences in distribution of CCL4 and CCL3 encoding genes and protein production, and suggest an intricate regulation of the CCL4L encoding genes. In order to investigate the role of CCL4 in HIV-1 control, we next assessed variation in the numbers of CCL4L copies in relation to CCL4 production in a cohort of 14 long-term nonprogressors (LTNPs). While no associations between copy number and CCL4 production were observed, the LTNP cohort had significantly lower levels of CCL4 production than the HIV-1-uninfected cohort, and this was maintained when Black and Caucasian individuals were examined individually. When the LTNP cohort individuals were divided based on viral loads, individuals with viral loads <400 RNA copies/ml had significantly lower CCL4 production than those with viral loads >400 RNA copies/ml. This finding suggests a role for the amount of CCL4 produced in the reduced pace of HIV-1 progression observed in LTNP individuals. Since genetic variation other than copy number can also influence CCL4 protein production, we then proceeded with a thorough genetic characterization of the CCL4 gene of uninfected individuals and investigated any possible relationships with select variants and CCL4 production. Sequencing the complete gene and flanking regions of 23 Black and 32 Caucasian revealed several intra as well as extragenic SNPs, with one newly identified SNP at position -1063. The Black population exhibited a higher degree of variability compared to the Caucasian population. We described four haplotypes in the Caucasian population, three haplotypes in the Black population, and a single haplotype shared between both population groups. Of the four indels that were identified, a three bp deletion (rs3216921) was the only indel present in the Caucasian population and was not identified in the Black population. Of all the indels, this indel had the highest allelic frequency (14%). Comparisons of haplotypes and prevalent SNPs with protein production in both population groups did not show any significant differences. Caucasian individuals harbouring the 3bp deletion however, had significantly higher levels of CCL4 production (p=0.024). These results form a good base from which to further investigate the impact of select genetic variants on CCL4 production and possibly HIV-1 control. This study has succeeded in optimising a ddPCR assay for the copy number determination of the CCL4 genes and has interrogated the relationship between CCL4L copy number and CCL4 production in both HIV-1-uninfected individuals as well as a subset of LTNPs. The results suggest a complex, intricate regulation of CCL4 that appears to play a role in HIV-1 control. In conclusion, this study forms the basis for future work to build on and to further explore the role of this CCR5 ligand in HIV-1 disease.

Research Report submitted to the School of Public Health University of the Witwatersrand, Degree of Master of Public Health 25 May 2015 / In Zimbabwe, HIV prevalence is higher among women than men of reproductive age- 18% vs. 12%. Gender power imbalances exist in our societies and result in relationship power imbalances; which increase women’s vulnerability to HIV. The aim of this study was to determine the correlation between relationship power and HIV sero-status among low-income urban Zimbabwean women attending post-natal care clinics. Methodology: A secondary data analysis of a cross-sectional survey conducted among 2 042 women aged 15-49 years, attending six low-income urban clinics in Harare in 2011. HIV results were based on rapid HIV diagnostic tests conducted during ANC. Shona intervieweradministered structured-questionnaires were used to collect data. This secondary analysis was limited to women with a known HIV status (n= 1 951). Multivariable logistic regression analyses were performed. Results: The study population mean age of the 26 years (SD=5.8). HIV prevalence was 14.6% (n=299). Having a partner who ever refused use of any family planning method was associated with the women’s HIV status (aOR 1.75, 95% CI 1.10-2.78). A non-significant association was found between relationship control by the male partner and women’s HIV status after adjusting for other factors. Conclusion: Although there were patterns of high male partner control in intimate relationships, not all women were without agency. Our study provides further evidence that male dominance in intimate relationships increases women’s vulnerability to HIV. HIV prevention programmes, interventions and policies should address gender issues to help curb this disproportionate pandemic among the woman sub-population.

One of the greatest barriers to curing HIV-1 is the ability of the virus to establish a state of latent infection within infected cells. During viral latency, the virus lies dormant in the form of an integrated, replication-competent provirus within the infected cell. In this state, the virus is undetectable by the host immune system and is unaffected by antiretroviral drugs due to extremely weak or null transcription. These viruses, however, can be induced to produce infectious virus later on and propagate the infection as well as reseed the latent reservoir. The factors that lead to the establishment and maintenance of HIV-1 latency are not all known. Current latency reversal methods are unable to effectively purge the latent reservoir in HIV-1-infected patients, which begs the question whether there are populations of latently-infected cells that are not being targeted by present therapeutics. One of our first lines of defense against retroviral infection is the APOBEC3 family of cytidine deaminases. These enzymes restrict retroviral infection mainly through hypermutation of the proviral DNA, leading to inactivation of the virus. However, it has been shown that low levels of mutations do not necessarily inactivate the virus and can sometimes be beneficial for the virus by increasing genetic diversity and viral fitness. Sublethal mutagenesis has been studied on coding regions of the virus, however, their effects on regulatory regions of the virus such as the LTR promoter have been scarcely explored. My project aimed to examine the effects of APOBEC3-induced mutations on the activity of the HIV-1 LTR promoter and investigate whether these mutations could be generating a subset of latently-infected cells. A library of HIV-1 clones with A3G- or A3F-mutated LTRs was generated. We discovered that the 5’ LTR is not mutated during the first round of HIV-1 infection in our system while the 3’ LTR accumulates mutations. A second round of infection is required for the mutations in the 3’ LTR to be copied over to the 5’ LTR and influence promoter activity. The mutations generated a range of effects on the promoter, with some clones being completely inactivated and no longer responsive to Tat or PMA/Ionomycin induction, while other clones were still highly infectious. The clones of interest were the ones in between this spectrum that were weakly-infectious (≤0.25%) prior to stimulation but were able to be induced above a given threshold (≥10-fold). We denoted these clones latency-prone viruses (LPVs) and were able to identify 10 of these clones within our library. We also explored the effects of individual mutations on the promoter and although these clones were highly infectious, we identified 8 positions of interest that led to weaker infectivity and fluorescence when mutated. The mutations in our library were found to hit important transcription factor binding sites and were also identified in a bioinformatics search of HIV-1-infected patient sequences, which confirms the relevance of our in vitro-identified mutations in a physiological setting. This is the first investigation and evidence of the APOBEC3 proteins contributing to the generation of a subset of latent viruses and constitutes an important contribution to the understanding of HIV-1 latency and its reversal. This previously uncharacterized pool of latently-infected cells could explain why current therapies are ineffective as they do not target these molecular modifications but rather focus on reversing epigenetics and reactivation of the virus. These newly-discovered latency-prone viruses would be a useful tool in testing reactivation drugs and establishes the need to develop novel antiretrovirals that will target a broader spectrum of latently-infected cells. This project has effectively illustrated the dual role of this host-encoded restriction factor and provided further insight into HIV-1 latency, the major hurdle towards a cure for HIV-1.

Thesis (M.Ed.) --University of Limpopo, 2004. / This study investigates how schools respond to and manage HIV and AIDS within the Limpopo Province of South Africa. Twenty-two stakeholders (parents, learners, HODs’, teachers and principals) and two government (provincial) officials participated in the present study. Three schools were selected by means of purposive sampling. Qualitative data were collected by means of two methods, namely, interviews and document analysis. By way of data processing and analysis, the responses from three schools in the Limpopo Province were categorized for the purpose of the study. The study found that stakeholders do not seem to understand issues related to HIV and AIDS, and that there are no proper mechanisms in place to deal with these issues. / University of the North

Thesis (MPH) --University of Limpopo, 2009 / The aim of the study was to determine the challenges of scaling up and strengthening quality-assured laboratory services for diagnosis and monitoring tests for HIV / AIDS patients on Anti- retroviral Therapy (ART). The objectives of the study were to: review the current national HIV/AIDS/STI/TB policy, Laboratory policy, ART strategic plan and guidelines on the implementation of ART services in Zambia; assess the knowledge, attitudes, and practices (KAP) of medical doctors/clinicians and knowledge and practices of laboratory staff in the diagnosis and monitoring tests for HIV / AIDS patients on ART; assess the quality of laboratory services for diagnosis and monitoring tests of HIV / AIDS patients on ART in Zambia compared to WHO standard guidelines; quantify the time taken for CD4 count results to reach the ART centres and determine the difference between the knowledge, attitudes and practices (KAPs) of medical doctors/clinicians in the ART centres with and without laboratory services for diagnosis and monitoring tests for HIV/AIDS patients on ART in Zambia. The study design was a cross-section descriptive survey of one hundred and thirty-seven (137) ART centres in the public health sector of the nine (9) provinces in Zambia. The study population consisted of six directors and managers from the Ministry of Health at national level, medical doctors/clinicians, laboratory staff, district directors of health, in charge of ART centres, and data-entry clerks in charge of Health Information Management Systems (HIMS) from one hundred and thirty-seven (137) ART centres in the public health sector in Zambia. The study findings indicated that only 23% of public sector laboratories were offering a full complement package of quality-assured laboratory services to support the ART programme in Zambia. The HIV/AIDS policy, Laboratory policy, Laboratory Standard Operating Procedures (SOPs) and guidelines on ART scale-up implementation plans exist at national level but had not been fully disseminated to all the ART centres. The average number of qualified laboratory staff at district hospitals surveyed was one (1) qualified laboratory personnel which is lower than the WHO recommendation of four (4) staff per district hospital. Most of the laboratories had no CD4 count machines to support ART V services. Unfortunately, CD4 count results took more than a week to reach the ART centres. Laboratories surveyed indicated a lack of equipment maintenance plans and service contracts. External Quality Assessment for diagnosis and monitoring tests for HIV/AIDS patients on ART was not yet well established. The findings also indicated that Medical Doctors/Clinicians working in the ART centres with laboratory services to support ART programme had better prognosis and treatment of patients on ART compared to those working in the ART centres without laboratory services. There was no difference in the knowledge, attitude and practices of Medical Doctors/Clinicians in the diagnosis and monitoring tests for the management of HIV/AIDS patients on ART in ART centres with and without laboratory service to support the ART programme in Zambia. In conclusion, the Ministry of Health should improve and increase accessibility to fully functional laboratory services to support ART programmes in order to reduce turn-around time for the CD4 count results to reach the ART centres. CD4 count machines should be provided to all the laboratories in ART centres and include service maintenance contracts to support ART services. The policy and decision makers should improve and strengthen the quality of laboratory services by disseminating the National HIV/AIDS policy, Laboratory policy, Laboratory SOPs and guidelines on ART scale-up implementation plan. The recruitment, training and improvement of redistribution of qualified staff should be accelerated to accommodate the current high workload, range of tests performed and an increase in laboratory operations with ART scale-up programme. A standard format of recording and reporting CD4 count results should be put in place (i.e. computerised or manual system). The Ministry of Health should develop guidelines and establish quality assurance systems and affiliate the laboratories to participate in the &ADC regional External Quality Assurance for accreditation such as the South African National Accreditation Systems (SANAS), to support the ART programme.