Introduction. The HIV prevalence among men who have sex with men (MSM) in China keeps increasing sharply. A high proportion of the MSM in China have male regular sex partner (RP) and prevalence of unprotected anal intercourse (UAI) involving such RP is higher than when non-RP is involved. Trust, intimacy and cognitive factors are the factors associated with UAI with RP. Several cross-sectional studies have been demonstrated the important factors associated with UAI with RP among MSM in China. However, no study about intervention for MSM-RP is found to be conducted. To reduce UAI with RP, an intervention tailored to RP is urgently developed and identified its efficacy. / Objectives. The study aimed to describe the prevalence of UAI, as well as of which associated factors among MSM-RP in Beijing and Chengdu, China, and to evaluate the efficacy of an enhanced HIV voluntary counseling and testing (VCT) in increasing condom use with RP among MSMRP in China by a randomized controlled trial (RCT). / Subjects and Methods. A cross-sectional survey and a randomized controlled trial have been conducted. For the cross-sectional survey, total 307 HIV negative MSM who have RP have been recruited by three ways. Face to face interview has been conducted to participants. Based on the associated factors found in the cross-sectional survey, interventions including video, education leaflets and enhanced counseling contents have been tailored to RP among MSMRP. For the randomized controlled trial, total 336 MSMRP have been recruited and randomly assigned 169 subjects to the Intervention Group in which participants have been given enhanced VCT plus an audio-visual and four leaflets components and 167 subjects to the Control Group in which participants have been given only standard-of-care VCT at the baseline. Evaluation was conducted at Month 3 and 6. Statistical methods such as descriptive analyses, Chi-square test and logistic regression were used in this study. / Results. The results have been found were the prevalence of UAI with RP among MSMRP was 52.4%, and the Theory of Planned Behavior (TPB) related cognitions, trust, intimacy, depression and anxiety were associated with UAI with RP among MSMRP. In the RCT study, participants in the Intervention Group had less UAI (36.1% vs. 49.1%) than those of the Control Group at Month 3. / Conclusions. This study showed a high prevalence of UAI among MSMRP, whilst trust, intimacy and cognitive factors were associated with UAI with RP. The efficacy of Enhanced VCT tailored to RP has been identified. The acceptability and feasibility of the tailored intervention were demonstrated. In the future HIV prevention programs, the effective intervention should be considered to be incorporated into standard-of-care VCT procedures and be implemented in the specific population. / 介紹：中國男男性接觸者中的愛滋病發病率一直保持著上升的狀態。而在中國男男性接觸者中有很大比例存在著固定性伴侶。男男性接觸者同固定性伴發生無保護肛交行為的比例大於其同非固定性伴。信任，親密以及認知因素已經被證實是影響男男性接觸者同其固定性伴發生無保護肛交行為的因素。但是在中國還沒有發現專門針對有固定性伴的男男性接觸者的干預研究。為了降低男男性接觸者同其固定性伴的無保護肛交的發生率，針對有固定性伴的男男性接觸者的干預方法應該被發展同時證實其有效性。 / 目的：本研究目的在於調查北京及成都男男性接觸者的固定性伴的比例，及其影響因素，包括健康行為理論的影響因素以及人際關係因素。同時，本研究也驗證了以隨機對照實驗來評估針對有固定性伴男男性接觸者的提高型愛滋病自願檢測諮詢對減少其高危性行為的效果。 / 對象與方法：本研究由橫斷面研究以及隨機對照試驗組成。在橫斷面調查中，307名愛滋病陰性的有固定性伴的男男性接觸者被招募。基於在橫斷面調查中發現的對男男性接觸者與固定性伴間發生無保護性行為的影響因素，一項專門針對有固定性伴的男男性接觸者的提高型愛滋病自願檢測諮詢干預方法被發展應用了隨機對照試驗中已驗證其有效性。在隨機對照試驗中，169名和167名研究對象被招募並分別被隨機分配到干預組（接受提高型愛滋病自願檢測諮詢）和對照組（接受標準型愛滋病自願檢測諮詢）中。分別於干預後的3個月和6個月回訪進行干預結果的評估。在本次研究中，運用了卡方检验和logistic回歸等統計學方法。 / 結果：在橫斷面調查中發現，男男性接觸者同固定性伴的無保護肛交發生率為52.4%。影響與固定性伴無保護肛交的因素包括：健康行為理論（TPB）相關的認知，信任，親密以及抑鬱和焦慮。在隨機對照試驗中發現，在3個月隨訪中干預組的男男性接觸者與固定性伴發生無保護肛交的比例較對照組明顯降低（36.1% vs. 49.1%）。 / 結論：本研究結果顯示中國男男性接觸者的固定性伴的比例很高，同時幾乎一半的有固定性伴的的男男性接觸者同時有多個性伴，這就有增加感染愛滋病及其他性病的風險。一項針對有固定性伴的男男性接觸者設計的提高型愛滋病自願檢測諮詢方法在降低其危險性行為上的可接受性和有效性已經被證實。提示在今後的愛滋病干預項目中可以進一步的推廣應用。 / Li, Chunrong. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 149-178). / Abstracts and appendixes also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Li, Chunrong. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Thesis (M. Med. Community Health)--University of Pretoria, 2003. / Includes bibliographical references (leaves 73-77).

The present study explores Rhodes students' perceptions and attitudes towards HIV/Aids issues. This study focuses on risk behaviour, stigmatisation, social perceptions and voluntary counselling and HIV testing (VCT). There is a lack of research on student attitudes, knowledge and behaviour at Rhodes University. It was therefore deemed pertinent to research this topic in that context. It was envisaged that the study would provide insights to be used in the formulation of improved strategies for HIV/Aids programs and education, ultimately impacting on the exponential increase of the pandemic in the Southern African region. A sample of six hundred and seventy five Rhodes University undergraduates completed a survey and its findings were interpreted in terms of relevant literature. A mixed methods approach using qualitative and quantitative methods was used. A focus group consisting of seven post-graduate students informed the development of the survey along with relevant literature. Four departments from the faculties of Commerce, Humanities, Science and Law were randomly sampled for the survey phase. Statistica was used to calculate descriptive statistics while the chi-square statistic was applied to examine the relationships between the variables. The findings show that the majority of students have high intention levels in planning to use preventative behaviour. However, in practise, this may not be the case. Many students feel that they belong to high or medium risk groups, as opposed to the low-risk groups. In terms of motivation levels, only sixty three percent of students are highly motivated to protect themselves from HIV/Aids and one third of respondents felt that they could not ask their partner to accompany them for an HIV/Aids test. In addition, students who had received VCT were more likely to be positive about the counselling process.

Against the backdrop of the growing problem of AIDS orphans in South Africa and greater sub-Saharan Africa, this qualitative enquiry examines the lives of three South African orphaned children living with HIV / AIDS in a children's home in Cape Town. It aims to generate rich, child-centred descriptions of some of the significant experiences of the children's lives. Drawings, dialogue and narrative were employed to generate the primary data. This was supplemented by collateral interviews and other relevant records, e.g. medical and biographical. Existential-phenomenological theory informed the approach to data collection and analysis. Each child produced a series often to twelve impromptu drawings over a period often weeks. These drawings and transcripts of the children's verbal descriptions of their drawings were extensively analysed. Significant themes for each participant as well as themes common to all three were identified. Some of the central themes emerging include loss, abandonment, death, disease awareness and coping. The children's ability to develop adaptive coping mechanisms and resilience in the face of traumatic loss and terminal illness was a particularly outstanding feature of the findings. Recommendations are made regarding future research to address the lack of qualitative, child-focused investigations as well as appropriate interventions for addressing the psychosocial needs of orphaned children living with HIV/AIDS.

Thesis (M. Tech.) - Central University of Technology, Free State, 2010 / Early detection of mother-to-child transfer of Human Immunodeficiency Virus (HIV-1) is of the utmost importance for monitoring the success of intervention strategies, as well as for optimal treatment of HIV-positive children. Serology can only be used confidently after 18 months, as remaining antibodies from the mother may give false positive results. This leaves only molecular methods for early detection of the virus; unfortunately, the technology is still too expensive for general use. The aim of this project was to develop and validate a cost-effective, fast, early detection method for HIV infection in infants. PCR was chosen as the developmental method, a technique that amplifies proviral sequences of HIV DNA, detecting HIV infection in peripheral blood mononuclear cells (PBMC) from infants of seropositive women during neonatal (age less than 28 days) and post-neonatal periods. A method based on the commercial Roche HIV-1 DNA assay was chosen for implementation on the Roche LightCycler instrument. The published primer set was used to detect both HIV-1 DNA and an internal control. The target DNA for use as internal control was constructed from the plasmid pBR322 so that an AT-rich part of the plasmid was flanked by the HIV-1 primer-binding sites. The resulting amplicon was cloned into a vector and multiplied in E. coli. Amplification of the plasmid by PCR in the Roche LightCycler in the presence of SYBR Green created an amplicon having a Tm different (81 ± 1°C ) from that of the HIV-1 amplicon (84 ± 1°C) so that post-amplification melting can be used to differentiate between HIV-1 and internal control. After construction of the internal control, the reaction conditions were optimised so that the internal control would amplify strongly only in the absence of HIV-1 target DNA. Then 50 previously tested patient samples were analysed using the assay developed here. Only half of the known positive samples came up positive in the assay, indicating that it is not sensitive enough for diagnostic use in its current form. Various ways of improving the sensitivity are suggested for further development of the assay as described here.

The study sought to determine the meaning and interpretation by facility managers and nurses on utilisation of expanded programme on immunisation and integrated management of childhood illnesses (EPI and IMCI) programmes for follow-up and antibody testing of HIV-exposed infants (HEI) at 18 months. Also to understand the factors within the health systems that influence the follow-up and antibody testing. The study setting selected was six facilities in Steve Tshwete subdistrict in Nkangala district of Mpumalanga province in South Africa. The study used a hermeneutic phenomenology using in-depth interviews for collecting data from 4 facility managers and 12 nurses. The major themes that emerged from the interviews were referral, defaulting, integration, stigma, and off-site ART initiation within the health system. These were found to influence the utilisation of HEI and IMCI services for follow-up and management of HEI. It was also found that the importance of integrating the management of HEI into the EPI and IMCI cannot be overemphasised. It was concluded that the Health Department needs to be vigilant and use all available resources to manage HEI to meet the MDG 4 of prevention of infant mortality / Health Studies / M.A. (Nursing Science)

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: In recent years, HIV-associated cancers have proven to be the bane of our time, since HIV is decimating humanity across the globe, even in the twilight of the last century. Cancer rates continue to rise in developing countries, where 95% of the world’s HIV-infected population lives, yet less than 1% have access to antiretroviral therapy. HIV-infected individuals have a higher proclivity to develop cancers, mainly from immunosuppression. An understanding of the immunopathogenesis of HIV-related cancers (HRC) is therefore a major prerequisite for rationally developing and/or improving therapeutic strategies, developing immunotherapeutics and proplylatic vaccines. In this study, we explore the pathology of HIV-related cancer malignancies, taking into account the pathogenic mechanisms and their potential for improving the treatment of management of these malignancies especially in developing countries. We mathematically model the dynamics of malignant tumors in an HIV-free environment, investigate the impact of cancer malignancies on HIV-positive patients and explore the benefits of various therapeutic intervention strategies in the management of HIV-related cancers. We present two deterministic models of infectious diseases to implement these, and they were analysed. We use HIV-related lymphomas in the Western Cape of South Africa as a case study. We validated the proposed models using lymphoma incidence data from the Tygerberg Lymphoma Study Group (TLSG), Tygerberg Hospital, Western Cape, South Africa. We show that the increasing prevalence of HIV increases lymphoma cases, and thus, other HIV-related cancers. Our models also suggests that an increase in the roll-out of the HAART program can reduce the number of lymphoma cases in the nearest future, while it averts many deaths. Furthermore, the results indicate that a highly crucial factor to consider in the prognosis of the incidence of lymphoma (and other cancer types) in HIV-infected patients is their CD4 cell count, irrespective of whether the patient has developed an HRC or not.

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In 2007, AIDS caused an estimated 2.1 millions deaths worldwide; about 70% in sub-Saharan Africa. HIV preferentially targets activated CD4 T cells, expressing the major HIV receptor CD4, as well as the major chemokine coreceptors CCR5 and CXCR4. These coreceptors play a prominent role during HIV cell entrance phase, HIV transmission and also disease progression. They have been found to be differentially expressed by CD4 T cell subsets. Tuberculosis coinfection may enhance immune activation in vivo thus accelerating HIV disease progression and has become a major challenge in the control of TB in Africa. Introduction of HAART has reduced disease progression to AIDS, as well as risk of further morbidity and mortality. HAART results in a rapid decline of viral load and an initial increase of peripheral CD4 count, however little is known on the effect of HAART in regulation of coreceptor expression, immune activation status and CD4 T cell subset distribution in HIV infection and HIV/TB coinfection. This study is a cross-sectional analysis of coreceptor expression, immune activation status and CD4 T cell subpopulation distribution in South African HIV and HIV/TB coinfected patients before and after ARV. A total of 137 South African individuals were investigated, comprising 15 healthy normal donors (healthy subgroup), 10 patients with active pulmonary tuberculosis (PTB subgroup), 33 HIV-1 positive patients without active PTB (HIV subgroup), 23 positive patients with active PTB (HIV/PTB subgroup), 36 HIV-1 positive patients on ARV (HIV on ARV subgroup) and 20 HIV-1 positive patients with active PTB on ARV (HIV/PTB on ARV subgroup). CD4 absolute count and plasma viral load were determined for all donors. Freshly isolated PBMC were classified by flow cytometry into the following CD4+ T lymphocyte subsets: naïve (CD45+, CD27+), effector memory (CD45-, CD27-), central memory (CD45-, CD27+), and effector (CD45+, CD27-). Coreceptor expression and activation status was assessed by CCR5, CXCR4 and CD38 expression on CD4 T cell subsets. HIV, TB and HIV/TB coinfection was associated with a decrease in percentage CCR5+ T cells as compared to healthy controls, with the HIV/TB group showing the most extensive decrease. In treatment naive patients, CD4 T cells showed elevated surface expression of CCR5 and CD38 as determined by mean fluorescence intensity in HIV/TB co-infection compared to HIV infection alone. The percentage of antigen-experienced cells was higher in the HIV/TB co-infected group compared to the HIV group. The percentage of naïve T cells was decreased in both the HIV infected and the HIV/TB co-infected groups compared to healthy controls. HIV patients with more than 6 months of ARV showed decreased CCR5 and CD38 surface level expression in the HIV and the HIV/ TB co-infected subgroups. An increased percentage of naïve T cells was observed in the HIV infected subgroup, but not in the HIV/TB subgroup, similarly, a decreased percentage of antigen-experienced cells was observed in the HIV subgroup, but not in the HIV/TB co-infected subgroup. A positive correlation was found between CCR5 and CD38 expression, and CXCR4 and CD38 expression (Spearman coefficient of correlation respectively: r=0.59, p<0.001 and r=0.55, p<0.001). Furthermore we found plasma viral load positively associated with CD38 expression (r=0.31, p<0.001) and percentage activated CCR5+ expressing CD4 T cells positively related to viral load (r=0.31, p<0.001). Percentage naïve CD4 T cells was positively associated with CD4 count (r=0.60, p<0.001) and negatively correlated to viral load (r=-0.42, p<0.001). These results indicate that TB coinfection exacerbates certain aspects of dysregulation of CD4 T cell homeostasis and activation caused by HIV infection. In addition, ARV-associated decrease in coreceptor expression, immune activation status and a normalisation of CD4 T cell subset distribution was observed in HIV infected individuals, but not in HIV/TB coinfection. Despite viral suppression after ARV treatment, the decline in the immune activation marker CD38 and coreceptor CCR5 expression, increase in percentage naïve CD4 T cells and decrease of antigen-experienced cells did not reach the levels displayed in the healthy control group. This may indicate that ongoing (albeit reduced) T cell immune activation may occur in the presence of ARV. Further longitudinal studies are needed to closely monitor immune activation during ARV treatment. This study highlighted an association of TB disease with immune activation in HIV infection, the importance of T-cell activation in HIV pathogenesis and its impact on ARV treatment. Further studies are needed to identify causative factors that may lead to a persistent immune activation status during ARV treatment, and how TB coinfection confounds normal responses to ARV. / AFRIKAANSE OPSOMMING: In 2007 was ongeveer 2.1 miljoen sterftes wêreldwyd veroorsaak deur VIGS; ongeveer 70% in Sub-Sahara Afrika. CD4 T selle is die hoof teiken van MIV, aangesien dit die primêre CD4 reseptor, sowel as een of beide van die vernaamste chemokien koreseptore CCR5 en CXCR4 vrystel. Hierdie koreseptore speel ‘n prominente rol wanneer die MIV die sel binnedring, asook tydens MIV oordrag en verloop van die siekte. Dit word ook deur verskillende fraksies van CD4 T selle vrygestel. Gelyktydige TB infeksie mag immuunaktivering in vivo verhoog en dus die siekeproses versnel. MIV het ‘n groot uitdaging geword in die beheer van TB in Afrika. Bekendstelling van HAART het die ontwikkeling van VIGS vertraag, asook die risiko van verdere morbiditeit en mortaliteit. HAART veroorsaak ‘n vinnige afname in virale lading ‘n toename in CD4 telling, hoewel die spesifieke invloed van HAART op die regulering van koreseptor vrystelling, immuunaktivering en verspreiding van CD4 fraksies in MIV en MIV/TB infeksies nog onduidelik is. Hierdie studie het gepoog om koreseptor vrystelling, immuunaktiveringstatus en die verspreiding van CD4 subpopulasies in pasiënte met MIV en MIV/TB voor en na ARV behandeling te ondersoek. ‘n Totaal van 137 Suid-Afrikaanse individue is ondersoek en die studiegroep het bestaan uit 15 normale persone (gesonde subgroep), 10 pasiënte met aktiewe pulmonale TB (PTB subgroup), 33 MIV positiewe pasiënte sonder PTB (MIV subgroep), 23 MIV positiewe pasiënte met aktiewe PTB (MIV/PTB subgroep), 36 MIV positiewe pasiënte op ARV (MIV op ARV subgroep) en 20 MIV positiewe pasiënte met aktiewe PTB op ARV (MIV/PTB op ARV subgroep). Absolute CD4 telling en virale ladings was bepaal vir alle deelnemers. Vars geïsoleerde perifere bloed mononukleêre selle is geklassifiseer deur middel van vloeisitometrie as die volgende CD4 T limfosiet subgroepe: naïewe selle (CD45+, CD27+), effektor geheueselle (CD45-, CD27-), sentrale geheueselle (CD45-, CD27+), en effektor selle (CD45+, CD27-). Koreseptor vrystelling en aktivering was beoordeel volgens CCR5, CXCR4 en CD38 vrystelling op CD4 T sel subgroepe. HIV, TB en MIV/TB ko-infeksie is geassosieer met ‘n afname in die persentasie CCR5+ T selle, vergeleke met gesonde kontroles, waar die MIV/TB subgroep die grootste afname getoon het. In onbehandelde pasiënte het die CD4 T selle verhoogde vrystelling van CCR5 en CD38 op die oppervlakte getoon en dit is bevestig deur die gemiddelde fluoresserende vii intensiteit in die MIV/TB subgroep vergeleke met die subgroep met slegs MIV. Die MIV/TB subgroep het verder ook ‘n verhoogde persentasie totale geheue T selle getoon vergeleke met die MIV subgroep. Die persentasie naïewe T selle was egter verlaag in beide die MIV en MIV/TB subgroepe vergeleke met normale kontroles. MIV pasiënte wat langer as 6 maande op ARV behandeling was in beide die MIV en MIV/TB subgroepe, het ‘n verlaagde vrystelling van CCR5 en CD38 op die oppervlakte van die CD4 selle getoon. ‘n Verhoogde persentasie naïewe T selle het in die MIV subgroep voorgekom, maar nie in die MIV/TB subgroup nie. ‘n Soortgelyke tendens is gevind waar die persentasie totale geheueselle verlaag was in die MIV subgroep, maar nie in die MIV/TB subgroep nie. ‘n Positiewe korrelasie is gevind tussen CCR5 en CD38 vrystelling, asook CXCR4 en CD38 vrystelling (Spearman korrelasie koëffisiënt: r=0.59, p<0.001 en r=0.55, p<0.001 onderskeidelik). Verder het die plasma virale lading ‘n positiewe assosiasie getoon met CD38 vrystelling (r=0.31, p<0.001) en die persentasie geaktiveerde CCR5+ vrystellende CD4 T selle met virale lading (r=0.31, p<0.001). Die persentasie naïewe CD4 T selle het ‘n positiewe assosiasie getoon met CD4 telling (r=0.60, p<0.001) en ‘n negatiewe korrelasie met virale lading (r=-0.42, p<0.001). Volgens hierdie resultate vererger TB ko-infeksie sekere aspekte van die disregulasie van CD4 T selhomeostase en aktivering as gevolg van MIV infeksie. Verder kon ‘n ARVgeassosieerde afname in koreseptor vrystelling, immuunaktivering en normalisering van CD4 T sel fraksies bespeur word in die MIV subgroep, maar nie in die MIV/TB subgroep nie. Ten spyte van virale onderdrukking veroorsaak deur ARV behandeling, het die afname in die immuunmerker CD38 en koreseptor CCR5, toename in die persentasie naïewe CD4 selle en afname in totale geheue CD4 T selle nie die vlakke van die normale kontrolegroep bereik nie. Dit is moontlik dat volgehoue verlaagde T sel immuunaktivering nog steeds mag plaasvind in die teenwoordigheid van ARV. Verdere longitudinale studies is nodig om immuunaktivering tydens ARV behandeling te monitor. Hierdie studie het die belangrikheid van T sel aktivering in MIV patogenese en dit impak daarvan op ARV behandeling beklemtoon. Verdere studies is nodig om moontlike oorsake of bydraende faktore te identifiseer wat tot volgehoue immuunaktivering tydens ARV behandeling kan lei, asook tot mate waartoe TB ko-infeksie kan inmeng met die normale werking van ARV behandeling.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / It is estimated that 32.8 million people are living with Human Immunodeficiency Virus (HIV) globally with the number of people receiving antiretroviral therapy in low- and middle- income counties increasing to more than 5 million people in 2009. These successes are threatened by treatment failure and the development of resistance to treatment. With an estimated 3.7% patients failing first line treatment after 2 years and 17.9% after 4 years on treatment there is a need for a practical and cheap in-house drug resistance assay that can be used to provide drug resistance data to clinicians and to use as a research tool to investigate drug resistance. In this study we attempted to optimize and validate an in-house drug resistance assay, adapted from Jacobs et al, 2008, to be used as a diagnostic tool and to study the presence of antiretroviral resistance in patients on the Western Cape Mother-To-Child-Transmission (MTCT) regimen. Quality control samples were received from The National Institute of Communicable Diseases AIDS Virus Research Unit, The Round Robin HIV-1 genotyping assessment system from the University of Würzburg and the QCMD assessment system were used for the optimization and validation of an in-house drug resistance assay. The ViroSeq™ HIV-1 Genotyping System was used for comparison of sample and mutation detection. It was possible to optimise and validate a genotyping assay for diagnostic testing and research use by comparison with the ViroSeq™ HIV-1 Genotyping System and evaluation with external quality assessment systems. This assay could subsequently be used to determine the development of genotypic-antiretroviral resistance in patients treated according to the provincial prevention of mother-to-child-transmission (PMTCT) protocol in the Western Cape (single dose nevirapine (sd-NVP), combined with a short course Zidovudine (AZT)). Patient samples were collected from pregnant women who took part in the Western Cape PMTCT program and visited the Tygerberg Obstetrics Clinic and Delft Community Hospital. EDTA blood was obtained to measure CD4-cell count, viral load, and to do genotyping for viral subtype and the presence of resistance mutations. Information on prior exposure to antiretroviral therapy was also collected. A detected resistance rate of 17.1% in this predominantly HIV-1 subtype C population is lower than previously recorded when sd-NVP was administered to HIV-1 subtype C positive patients in PMTCT programs. This could indicate that a dual PMTCT regimen including AZT and NVP reduces the risk of resistance to NVP relative to a regimen that uses sd-NVP. The genotyping assay uses four primers to amplify the PR and the RT gene separately to obtain PCR products, of 487 and 804 base pairs respectively for sequencing. The two PCR products were sequenced with three and five primers respectively to sequence the complete PR and approximately 250 amino acids of the RT gene. The sequences generated, thus, are analysed and aligned with the Sequencer V4.7 software to obtain a consensus sequence of approximately 1200 base pairs for analysis of resistance mutations in the protease and reverse transcriptase genes. The developed assay was hence further simplified and improved, by combining the PR and RT assay into one, which was optimised and validated for use in the routine diagnostic setting. The final genotyping assay uses 8 primers for sequencing to obtain a 1200 bp sequence for genotyping that contains the protease and the 5’ of the reverse transcriptase genes in which antiretroviral resistance associated mutations are found. The assay was accredited by SANAS in 2008.

Thesis (MScMedSc)--Stellenbosch University, 2013. / Bibliography / The efficiency in which HIV-1 can infect, spread and evade the attack of therapeutic agents can be attributed to a high mutation rate and frequent recombination events. These factors have collectively contributed to the diversity observed in HIV-1 and resulted in a multitude of subtypes, sub-subtypes, circulating recombinant forms (CRF’s) and unique recombinant forms (URF’s). The aim of this study was to investigate HIV-1 diversity in Cape Town using a small cohort of treatment naive patients being investigated for HIV Associated Neurocognitive Disorders (HAND). Four different genomic domains: gag, pol, accessory and gp41 genes were sequenced to subtype the virus. HIV-1 tat was further investigated because the dicysteine motif has been reported to play a role in HAND. Viral RNA and proviral DNA was extracted from 64 patients and used for the amplification and sequencing of the genes. Rega and jpHMM online tools were used to identify HIV-1 subtypes and recombinants while Neighbor-joining phylogenetic trees were constructed for phylogenetic analysis. The pol gene was further investigated using SCUEAL to detect possible intra-subtype recombination and was also screened for the presence of transmitted drug resistance. In addition tat sequence datasets retrieved from the Los Alamos sequence database were investigated and compared with the newly generated sequences for the detection of point mutations and amino acid signature patterns. Sequencing identified most of the samples as subtype C; however six inter-subtype recombinants (AE, A1G, A1CU and two BC) and 9 intra-subtype C recombinants were identified. In addition 13% of pol sequences were identified with resistance mutations. Signature pattern analysis identified a high level of variability in the tat sequences: 68% were identified with C30S31; 29% with the C30C31 mutation and a single sequence with a novel mutation C30A31. Functional analysis of these mutations indicated that all mutations investigated were capable of inducing apoptosis in cell culture. The C30C31 mutation generated the highest level of apoptosis, closely followed by the C30A31 mutation. However no statistical significance could be detected between tat mutations and the observed levels of apoptosis.