Thesis (Ph.D.(Public Health))--University of the Witwatersrand, Faculty of Health Sciences, 2012. / Measuring HIV incidence has proved challenging over the years. A number of serological HIV assays have been proposed, and among these, the BED Capture Enzyme Immunoassay (CEIA) is one of the more widely used. Although the assay performs well among known seroconverting panels, it has been shown to classify some long term infected patients as being recently infected. Information on the performance of the BED assay among low CD4 cell count patients and those on antiretroviral therapy is limited. The risk of onwards transmission of HIV has been reported to be elevated around the seroconversion period compared to the chronic stage of infection. RNA viral load has been reported as the strongest predictor of HIV transmission compared to other HIV markers. Understanding how these markers influence the relationship between the likelihood of being recently infected and the BED assay might help in understanding some of the shortcomings of the BED assay. The main aim of this study was to understand the properties of the BED assay. The performance of the BED assay among advanced HIV disease patients and the influence of ART on BED levels once patients started treatment was investigated. The BED assay and CD4 cell count were used to quantify the risk of in utero and intrapartum transmission to their infants among women believed to have seroconverted during pregnancy. The influence of viral load, haemoglobin and mid-upper arm circumference was investigated on the relationship between the probability of being recently infected and BED ODn levels. Methods Cryopreserved plasma samples from HIV patients on the national antiretroviral treatment (ART) rollout programme at Tygerberg Hospital HIV clinic, South Africa, iv were used to investigate the effect of ART on BED ODn levels once patients commenced treatment. Mixed effect logistic regression models accounting for multiple readings per patient were used. To investigate the risk associated with seroconversion during pregnancy HIV seropositive women who had just given birth were classified into mutually exclusive groups according to their likelihood of having recently seroconverted using BED and CD4 cell count levels. Multinomial logistic regression models adjusting for other factors were used to assess the risk of MTCT in utero and intra-partum infection comparing these groups. To investigate the relationship between BED ODn levels and the probability of being recently infected, BED data from known HIV infected women and women who seroconverted over a 2 year period was used. Fractional polynomial regression models that allow for non-linear functions to be fitted were used, and the influence of viral load, haemoglobin and mid-upper arm circumference was assessed through multi-variable models. Data from the Zimbabwe Vitamin A for Mothers and Babies (ZVITAMBO) project, a double blinded treatment-placebo trial was used for these last two objectives. Results Patients with very low CD4 cell counts were more likely to test false recently infected according to the BED assay than other patients. ART changed BED ODn kinetics among HIV patients on treatment. Over half of advanced disease stage patients were likely to be classified as being recently infected according to the BED assay 2 years into ART treatment. v Women who seemed to have seroconverted during pregnancy had elevated risk of transmitting HIV in-utero compared to chronic HIV patients. BED and CD4 cell count were not predictive of risk of intra-partum infections attributed to seroconversion during pregnancy. The relationship between the probability of being recently infected with HIV and BED ODn levels was described better using Fractional Polynomial regression models than using a linear model in BED ODn or a model in which the BED ODn was categorised. Viral load and haemoglobin were important independent predictors of incident infections. Conclusions If the BED assay is to be used for HIV incidence estimations patients on ART should be accounted for. The BED assay together with other HIV serological markers can be used as prognostic tools to assess the risk of HIV transmission. The risk of in-utero transmission of HIV is higher among women who seroconvert during pregnancy. Repeat HIV testing among pregnant women may help in identifying women who seroconvert during pregnancy, and these women will benefit from Prevention of Mother-to-Child transmission (PMTCT) programmes. It was found that additional markers such as viral load and haemoglobin did not alter the relationship between the probability of having been recently infected and BED ODn.

Introduction Voluntary Counselling and Testing (VCT) play a crucial role in the control and management of the HIV/AIDS epidemic. It is essential to understand the factors that influence the utilisation of VCT to improve implementation of measures that encourage VCT uptake. The purpose of this study was to determine factors affecting the utilisation of Voluntary Counselling and Testing (VCT) services for HIV/AIDS in Sowa, Botswana. Methodology A cross-sectional study was carried out by collecting data from 71 randomly selected participants residing in the community of Sowa, Botswana. Open and close ended questions were used. Relevant demographic data were collected from each respondent. Univariate and multivariate analysis was done using chi square test and logistic regression models through STATA11 statistical software. Results About half of the respondents were willing to utilise the VCT services. Willingness to utilise VCT was significantly associated with the respondents' choice of VCT centres, worry about confidentiality at VCT centres, and concern about their partners' being aware of their use of VCT. The expected reactions of their partner, family and community to the use of VCT by the respondents, in addition to the willingness of respondents to inform their partners the result of their HIV test, were other factors associated with the use of VCT. Multivariate regression showed that being "not worried" (AOR 33.48; 95CI 5.63 - 199.15) about confidentiality at VCT centres predicted the willingness to use VCT. In addition, not worried that their partners were aware they had utilised VCT (AOR 7.25; 95CI 1.69- 31.14), and readiness to inform their partners about the result of their HIV test (AOR 14.96; 95CI 3.74- 59.85), predicted the willingness of respondents to utilise VCT. Similarly, the expectation of a happy reaction from partner (AOR 47.02; 95CI 3.83- 577.11) and family (AOR 45.13 95CI 3.28-620.72) on being aware the respondent had used VCT, also predicted willingness to use VCT. Conclusion This study confirmed that stigma related to VCT use and confidentiality at VCT centres influence the utilisation of VCT services. Also, concern about partner's awareness that a respondent had used VCT and the expected response of partner, family, and community, were all important influencing factors to the utilisation of voluntary counseling and HIV testing services. These issues need to be addressed in order to increase VCT uptake among individuals and the community.

The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun. / text