Thesis (MSc in Medical Science)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Background: Para-aminosalicylic acid (PAS) is one of the first effective anti-tuberculosis agents and has become one of the principal second-line drugs to treat patients with an extended resistance spectrum. Despite being one of the oldest anti-tuberculosis drugs, little data is available regarding its pharmacokinetics, drug interactions, genetic factors and dosing regimens, especially for the relative new granular slow release PAS (GSR-PAS) preparation. Objectives The aim of the study was to investigate the pharmacokinetics, tolerability and safety of a single 8 g once- or 4 g twice-daily GSR-PAS dose in a multidrug- or extensively drug resistant tuberculosis (M/XDR-TB) population, in which some subjects were also co-infected with the human immunodeficiency virus (HIV). An additional objective was to investigate the potential covariates (i.e. genetic factors and drug interactions) that can alter the pharmacokinetics of PAS. Study design and methodology A randomised, two-period, open-label cross-over study was conducted in 32 adults (≥18 years old) with M/XDR-TB admitted at Brooklyn Chest Hospital, Cape Town, South Africa and treated for drug resistant tuberculosis with a multidrug regimen containing GSR-PAS. The subjects were randomised to follow a single 8 g once-daily GSR-PAS regimen or a 4 g twice-daily GSR-PAS regimen for 8 days. On the eighth day blood samples were obtained at 0, 1, 2, 3, 4, 6, 8, 12 and 24 hours. After the 24-hour sample (Day 9) the regimens were crossed-over. The tolerability and safety of the two regimens were determined using Visual Analogue Scales and interviews. PAS plasma concentrations were determined by a developed HPLC-MS/MS method. N-acetyltransferase (NAT1 and NAT2) genotyping was performed. The data of this study together with unpublished data of a previous study in a very similar population were used in a pharmacometric analysis to determine the PK parameters and any subject covariates. Results and Discussion In comparison to the 4 g twice-daily GSR-PAS dose, the single 8 g once-daily GSR-PAS dose generated a pharmacokinetic profile with a significantly higher maximum concentration (Cmax), concentration at 12 hours (C12) and area under the curve 0 to 12 hours (AUC12). The concentrations of all subjects on the twice-daily regimen were maintained above a minimum inhibitory concentration (MIC) throughout a 12-hour interval, while the single 8 g dose was able to sustain the PAS plasma concentrations above the MIC in 18 out of 29 subjects (62.1%) for the entire 24-hour dosing interval. Both regimens were reasonably well tolerated but most subjects preferred the twice-daily dosing. The clearance of PAS was increased by 45% in HIV positive subjects prescribed antiretroviral treatment (ART), possibly due to interaction with efavirenz (EFV). No significant associations were found for any of the individual NAT1 or NAT2 genotypes, but a difference between mean concentrations of the different genotypic groups was reported. Conclusions The 8 g once-daily dose has the potential to be included in future regimens. The higher peak concentrations achieved can be expected to increase the bactericidal effect of GSR-PAS without significant loss of bacteriostatic effect, i.e. time over MIC. The 8 g once-daily dose has a reasonable tolerability and is potentially easier to supervise in an outpatient setting. Since antiretrovirals (ARVs) increase PAS clearance and decrease PAS exposure in HIV co-infected subjects on ART, the 8 g once-daily dose will be less suitable for maintaining bacteriostasis in these patients (inadequate PAS exposure). Therefore, PAS and ARV interactions need to be clarified before the 8 g once-daily dose can be recommended for the HIV co-infected patients on ART. / AFRIKAANSE OPSOMMING: Agtergrond Para-aminosalisielsuur (PAS) is een van die eerste effektiewe anti-tuberkulose middels en het een van die hoof tweede-lyn middels geword om pasiënte met 'n uitgebreide weerstand spektrum te behandel. Ondanks die feit dat PAS die oudste anti-tuberkulose middel is, is daar baie min data beskikbaar met betrekking tot die farmakokinetika, middel interaksies, genetiese faktore en dosering, veral in die geval van die relatiewe nuwe granulêre stadige vrystelbare PAS voorbereiding (GSV-PAS). Doel Die doel van die studie was om navorsing te doen oor die farmakokinetika, verdraagsaamheid en veiligheid van ʼn enkele 8 g een keer daaglikse en 4 g twee keer daaglikse GSV-PAS dosering in ʼn multi- of uitgebreide weerstandige tuberkulose (M/XDR-TB) populasie, waar sommige proefpersone ook met die Menslike Immuniteitsgebreksvirus (MIV) geko-infekteer is. ʼn Verdere doel van die studie was om te bepaal of potensiële kovariate soos genetika en medisyne interaksies die farmakokinetika van PAS verander. Metodes ʼn Onwillekeurige, twee-periode, oop-etiket oorkruisingstudie was op 32 M/XDR-TB volwassenes (≥ 18 jaar oud) uitgevoer terwyl hulle vir middel weerstandige tuberkulose in Brooklyn Chest hospitaal (Kaapstad, Suid-Afrika) behandel is. Die deelnemers was onwillekeurig ingedeel om ʼn 8 g eenkeer daaglikse GSV-PAS dosering of ʼn 4 g twee keer daaglikse GSV-PAS dosering vir agt dae te volg. Op die agste dag was bloedmonsters op die volgende ure 0, 1, 2, 3, 4, 6, 8, 12, en 24 geneem. Na die 24-uur monster (Dag 9) was die doserings omgekeer. Die verdraagsaamheid en veiligheid van die twee doserings is bepaal deur gebruik te maak van Visueel Analogiese Skale en onderhoude. PAS plasma konsentrasies is bepaal deur 'n ontwikkelde HPLC-MS/MS metode. N-asetieltransferase (NAT1 en NAT2) genotipering is uitgevoer. Die data van hierdie studie saam met ongepubliseerde data van 'n vorige studie is gebruik in farmakometriese analise om die farmakokinetiese parameters en enige kovariate te bepaal. Resultate en Bespreking In vergelyking met die 4 g GSV-PAS twee keer daaglikse dosis, het die enkele 8 g daaglikse dosis, ‘n pharmakokinetiese profiel met ‘n beduidende hoër maksimum konsentrasie (Cmax), 12-uur konsentrasie (C12) en area onder die kurwe van 0 tot 12 uur (AUC12), gegenereer. Die PAS plasma konsentrasies van alle proefpersone, wat op die twee keer daaglikse dosis was, was tydens die 12-uur interval bo die die minimum inhiberende konsentrasie (MIK) gehou. Terwyl die enkele 8 g dosis die PAS plasma konsentrasies vir die duur van die 24 uur interval bo die MIK in 18 van 29 (62%) proefpersone gehandhaaf het. Die meeste proefpersone het die twee-daaglikse dosering verkies, maar beide doserings was redelik goed verdra. Die verwydering van PAS het met 45% toegeneem in HIV positiewe proefpersone wat antiretrovirale behandeling ontvang het, moontlik weens interaksies met efavirenz. Geen beduidende assosiasies vir enige van die individuele NAT1 of NAT2 genotiepes was gevind nie, maar ‘n verskil tussen die gemiddelde konsentrasies van die verskillende genotiepes is gerapporteer. Gevolgtrekking Die 8 g een keer daaglikse dosis het die potensiaal om in toekomstige doserings ingesluit te word. Die hoër piek konsentrasies van die 8 g daaglikse dosis, kan moontlik die bakterisidiese (kiem-dodende) effek van GSV-PAS verhoog, sonder om die beduidende bakteriostatiese (kiem-inhiberende) effek (o.a. tyd oor MIK), te verloor. Die 8 g een keer daaglikse dosis is redelik verdraagsaam en kan potensieël makliker gekontrolleer word in die geval van buite-pasiënte. Serdert antiretrovirale middels (ARVs) PAS verwydering verhoog en gevolglik die PAS plasma konsentrasies verlaag in die MIV ko-infekteerde proefpersone wat op ARVs is, sal die 8 g een keer daaglikse dosis minder geskik wees vir die handhawing van bakteriostasis in hierdie pasiënte (onvoldoende PAS blootstelling). Dus moet daar klarigheid verkry word oor PAS en ARV interaksies voordat die 8 g een keer daaglikse dosis vir MIV ko-infekteerde pasiënte op ARVs aanbeveel kan word.

Thesis (MA )--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The purpose of this thesis is to assess the impact of HIV and AIDS on democratic consolidation in two democracies in Southern Africa: Botswana and South Africa. Mattes (2003), Barnett and Whiteside (2006) and others warned that in states with high HIV infection levels, the negative impact of the pandemic - especially in terms of socio-economic conditions, budgetary pressures and a loss of human capital in the state and the economy - was potentially so great that it may affect democracy detrimentally. In contrast, some scholars, particularly Anthony Butler (2005a) and Alex de Waal (2006), contended that although the pandemic had negative effects, democracies might survive it and that in some specific ways, democratic consolidation might even benefit from the its consequences. For instance, they argued that in South Africa, the civil society response to the government’s controversial HIV and AIDS policy deepened the institutional framework of democracy. The methodology for the above comparative analysis is based on the application of a minimalist multivariate model which, following the thinking of Bratton and Van de Walle (1997) consists of both institutional and socio-economic factors. Factors are selected for their relevance to democratic consolidation, as argued by scholars such as Linz and Stepan (1996), Przeworski, Alvarez, Cheibub and Limongi (1996), Bratton and Van de Walle (1997) and Leftwich (2000). The chosen factors are the system of government (the relationship between the branches of government); the electoral system; political rights and civil liberties; economic indicators (affluence, economic growth and the reduction of inequality); human development (as measured by the United Nations Development Program) and civil society. This is a descriptive, qualitative, desktop study, using secondary literature in books, as well as articles. There is no empirical component, such as fieldwork, surveys or questionnaires. As stated below, such methodology may be used for further elaboration and refining of the findings of this desktop-based comparative analysis. The main finding is that currently, despite the cost and human implications of the disease, there are no indications that it is directly threatening to destroy the democracies of Botswana or South Africa. This finding differs from the more negative expectations of the scholars mentioned above. It is suggested that the increasing provision and effectiveness of antiretroviral treatment (ART) enables these democracies and their economies to avoid some of the ravages of the disease that seemed inevitable a few years ago. Furthermore, it is suggested that the comparative affluence of the two states in question shields them from some negative effects of HIV and AIDS and that this may be different in poorer Southern African states. This is an issue for further research. Such research should go beyond desktop research to include fieldwork and questionnaires. / AFRIKAANSE OPSOMMING: Die doel van hierdie tesis is om die impak van MIV en VIGS op demokratiese konsolidering in twee Suider-Afrikaanse demokrasieë, Botswana en Suid-Afrika, vas te stel. Mattes (2003), Barnett en Whiteside (2006) en ander het gewaarsku dat die negatiewe uitwerking van die pandemie - veral in terme van sosio-ekonomiese toestande, begrotingsdruk en ’n verlies aan menslike hulpbronne in die staat en ekonomie - potensieel so groot is dat dit demokrasie nadelig sou beïnvloed. In teenstelling hiermee het ander akademici, soos Anthony Butler (2005a) en Alex de Waal (2006), geredeneer dat demokrasieë die pandemie mag oorleef ten spyte van die negatiewe effekte wat dit wel het en dat demokrasieë selfs op sekere wyses by die gevolge daarvan mag baatvind. Byvoorbeeld, het hulle geargumenteer, in Suid-Afrika het die burgerlike samelewing se reaksie op die Mbeki-regering se kontroversiële MIV en VIGSbeleid die institusionele raamwerk van demokrasie verdiep. Die metodologie vir hierdie vergelykende analise is gebaseer op die toepassing van ’n minimalistiese multiveranderlike model. Soos gepostuleer deur Bratton en Van de Walle (1997), wat beide institusionele en sosio-ekonomiese faktore insluit. Faktore is gekies op grond van hulle relevansie tot demokratiese konsolidering (volgens vakkundiges soos Linz en Stepan (1996), Przeworski, Alvarez, Cheibub en Limongi (1996), Bratton en Van de Walle (1997) en Leftwich (2000), asook vir dié se moontlike relevansie tot demokrasieë wat spesifiek deur MIV en VIGS geaffekteer word. Die gekose faktore is die regeringstelsel (die verhouding tussen die uitvoerende, wetgewende en regsprekende gesag), die verkiesingstelsel, politieke regte en burgerlike vryhede, ekonomiese aanwysers (welvaart; ekonomiese groei en die vermindering van ongelykheid), menslike ontwikkeling (soos gemeet deur die Verenigde Nasies se Ontwikkelingsprogram) en die burgerlike samelewing. Hierdie tesis is ’n literatuurstudie van ’n beskrywende, kwalitatiewe aard. Daar is gebruik gemaak van sekondêre literatuur in boeke, asook van artikels. Daar is geen empiriese komponent soos veldwerk en meningspeilings nie. Soos hieronder beklemtoon word, kan empiriese metodes in toekomstige studies gebruik word om op die bevindinge wat hierdie navorsing opgelewer het, uit te brei en dit te verfyn. Die hoofbevinding is dat daar tans, ten spyte van die finansiële en menslike koste van MIV en VIGS, geen aanduiding is dat die siekte ‘n direkte bedreiging inhou vir die voortbestaan van demokrasie in Botswana en Suid-Afrika nie. Hierdie bevinding verskil van die meer negatiewe verwagtinge hierbo uitgespreek. Dit word voorgestel dat die toenemende voorsiening en effektiwiteit van antiretrovirale behandeling hierdie demokrasieë en hulle ekonomieë daartoe in staat stel om gedeeltelik die verwoesting van hierdie pandemie te vermy, iets wat enkele jare gelede nog as onvermydelik beskou is. Verder word die voorstel gemaak dat die impak van die pandemie op armer Suider-Afrikaanse state vergelyk behoort te word met die bevindinge wat hier aangebied word. Sulke toekomstige navorsing behoort nie net literatuurstudie in te sluit nie, maar ook veldwerk en meningsopnames.

Thesis (MSc)--Stellenbosch University, 2012. / Background: While Selection of reverse transcriptase (RT) mutation has been reported frequently, protease (PR) mutations on antiretroviral therapy (ART) including boosted Protease inhibitor (PI) have not been reported as much in Zambia. Affordable in-house genotyping assays can been used to expand the number of patients receiving drug resistance geno-typing, which can aid in determining prevalence of RT/PI emerging mutations. Methods: A previously published drug resistance genotyping assay was modified and used to genotype RT and PR genes. 19 patients virologically failing first-line regimen and 24 failing second-line regimen were studied to determine resistance patterns. Virological failure was defined as failing to maintain <1000 copies/mL during ART. Only major and minor RT and PR mutations (IAS-USA 2010) were considered for analysis. The in-house assay was validated by comparing sequence data of 7 previously ViroSeq tested samples and 5 randomly selected samples to determine reproducibility. Results: The in-house assay efficiently amplified all 12 validation samples with the lowest sample scoring 99.4% sequence homology. The most common RT mutation was M184V (79% n=19) and (71% n=24) first and second-line respectively. No significant differences were reported in all the other RT mutations between first-line and secondline regimens. Drug resistant PI mutations (I54V, M46I and V82A all present 20.8%) were only found in the second-line regimen and were insignificant, p= 0.0562. Conclusion: The in-house assays can be used as alternatives for commercial kits to genotype HIV-1C in Zambia without compromising test quality. The insignificant PI drug resistant mutations which were found, despite virological failure in patients, could indicate a possibility of other mutations within the HIV-1 genome that could reduce PI susceptibility.

Thesis (MSC)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: South Africa is shouldering the highest burden of HIV-infection. Inter-individual differences in response to antiretroviral treatment (ART) and the limited availability of second and third-line ART regimens call for optimising first-line ART in South African populations. Measuring antiretroviral drug levels in patients may be of clinical value as an intermediate indicator of treatment response and may moreover serve to assess the genetic variation underlying differential drug exposure. This study aimed to determine the effect of SNPs in the CYP2B6 gene and efavirenz (EFV) levels measured in hair on ART outcomes in females of two South African populations. Female Xhosa (XH) (n = 81) and Mixed Ancestry (MA) (n = 53) patients receiving the first-line regimen component EFV for at least three months donated saliva for genomic DNA extraction and 20 strands of hair for determination of EFV concentrations by high performance liquid chromatography. Regulatory and exonic regions in the CYP2B6 gene, which codes for the major metabolising enzyme of EFV, were subjected to bi-directional sequence analysis in 15 XH and 15 MA individuals to assess common genetic variation in these populations. Out of 45 single nucleotide polymorphisms (SNPs) identified, 17 SNPs of known or predicted functional importance in EFV metabolism, including four novel SNPs, were genotyped in the entire patient cohort by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. All SNPs were tested for Hardy-Weinberg equilibrium (HWE) and maximum likelihood haplotypes and assessed for an association with EFV levels measured in hair, likelihood of developing adverse drug reactions (ADRs) and virological response to EFV-based treatment. After correcting for age and ethnicity, homozygous carriers of c.516G>T (CYP2B6*6) had significantly increased EFV levels (p = 0.0021; mean: 12.0 ng/mg; IQR: 3.95 – 6.99 ng/mg; n = 12), as did heterozygotes of c.983T>C (CYP2B6*18) (p = 0.0005; mean: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). No CYP2B6*18 homozygotes were detected. No association between EFV levels and virological response was evident (p = 0.8467), but CYP2B6*6 predicted increased odds of virological failure (VL > 80 copies/ml) after correcting for adherence, race, age, weight, time on treatment, baseline CD4, smoking, alcohol and WHO disease stage (p = 0.0328). Carriers of the CYP2B6*1 allele had increased odds (OR = 5) of favourable treatment outcome (VL < 80 copies/ml). In accordance with other studies, this study provides evidence that genetically predisposed poor metabolisers of EFV may be at increased risk of virological failure, possibly following non-adherence. Concurrently, these patients may be more vulnerable to adverse drug reactions and are more frequent in the XH (13%) than MA (4%). These results should be verified in larger patient cohorts, but contribute to a better understanding of the effect of genetic factors on EFV exposure and ART outcome in two South African populations. The outcomes of this study may thus provide recommendations for prospective studies and impact future clinical decisions. / AFRIKAANSE OPSOMMING: Suid-Afrika dra die grootste las van MIV-infeksies. Inter-individuele verskille in reaksie op anti-retrovirale terapie (ART) en die beperkte beskikbaarheid van tweede- en derde-linie ART-reekse regverdig die optimisering van eerste-linie ART in Suid-Afrikaanse bevolkings. Meting van antiretrovirale middel-vlakke in pasiënte, as ‘n intermediêre aanduiding van reaksie op behandeling, kan van kliniese belang wees en kan ook die waarde van die bepaling van genetiese variasie, onderliggend aan differensiële blootstelling aan middels, bepaal. Die doel van hierdie studie is om die effek van enkel-nukleotied polimorfismes (SNPs) in die CYP2B6-geen en efavirenz (EFV)-vlakke in hare op ART-uitkoms te bepaal in vroue van twee Suid-Afrikaanse bevolkingsgroepe. Vroulike Xhosa (XH) (n = 81) en Gemengde Herkoms (GH) (n = 53) pasiënte wat EFV as deel van eerste-linie ART vir ten minste drie maande ontvang het, het speekselmonsters vir genomiese DNA-ekstraksie en 20 hare vir die bepaling van EFV-konsentrasies deur hoë werkverrigting vloeistofchromatografie (“HPLC”) geskenk. Regulatoriese en eksoniese areas in die CYP2B6-geen, wat vir die vernaamste metaboliserende ensiem van EFV kodeer, is deur middel van tweerigting-volgordebepalings-analise in 15 XH en 15 GH individue ondersoek om gemeenskaplike genetiese variasie in hierdie bevolkings te bepaal. Uit ‘n totaal van 45 SNPs wat geïdentifiseer is, is 17 SNPs wat bekende of voorspelde belangrike rolle in EFV-metabolisme speel, insluitend vier nuwe SNPs, ondersoek. Hierdie SNPs is in die volledige pasiënt-kohort gegenotipeer deur polimerase-ketting reaksie gebaseerde restriksie fragment lengte-polimorfisme (PKR-RFLP) analise. Alle SNPs is getoets vir Hardy-Weinberg-ewewig (HWE) en maksimum waarskynlikheidshaplotipes en is geassesseer vir assosiasie met EFV-vlakke gemeet in hare, die waarskynlikheid om ongunstige reaksies tot die middel te ontwikkel en virologiese reaksie op EFV-gebaseerde behandeling. Nadat vir ouderdom en herkoms gekorrigeer is, het homosigotiese draers van c.516G>T (CYP2B6*6) beduidend verhoogde EFV-vlakke (p = 0.0021; gemiddeld: 12.0 ng/mg; IQR: 3.95 – 6.99; n=12) getoon, so ook heterosigote vir c.983T>C (CYP2B6*18) (p = 0.0005; gemiddeld: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). Geen CYP2B6*18 homosigote is gevind nie. Daarbenewens is geen duidelike assosiasie tussen EFV-vlakke en virologiese reaksie gevind nie (p = 0.8467), maar CYP2B6*6 het verhoogde waarskynlikheid op virologiese mislukking (VL > 80 kopieë/ml) getoon nadat daar vir mddel-getrouheid, ras, ouderdom, gewig, tydsduur van behandeling, basis-CD4, rook, alkohol en Wêreld Gesondheids Organisasie siekte-fase gekorrigeer is (p = 0.0328). Draers van die CYP2B6*1-alleel het verhoogde waarskynlikheid (OR = 5) op gunstige behandelingsuitkomste getoon (VL < 80 kopieë/ml). In ooreenstemming met ander studies verskaf hierdie studie bewyse dat pasiënte wat geneties geneig is tot stadige metabolisme van EFV ‘n hoër risiko kan hê vir virologiese mislukking, wat moontlik ‘n gevolg is van middel-ontrouheid. Hierdie pasiënte kan ook meer geneig wees tot vatbaarheid vir ongunstige middel-reaksie en kom meer voor in die XH (13%) as in die MA (4%). Hierdie resultate moet in groter pasiënt-kohorte gestaaf word, maar dra by tot ‘n beter begrip van die effek van genetiese faktore op blootstelling aan EFV en ART-uitkoms in twee Suid-Afrikaanse bevolkings. Die uitkomste van hierdie studie kan dus as aanbevelings gebruik word vir voornemende studies en ook toekomstige kliniese besluite beïnvloed. / The Medical Research Fund (MRC) for funding this project. The University Centre for Studies in Namibia (TUCSIN) and Deutscher Akademischer Austausch-Dienst (DAAD) for financial support

HIV/AIDS is the most threatening and challenging infectious diseases of our time, with the highest increase of newly infected cases reported. This infectious disease was discovered in the early eighties under homosexual men and was later to be discovered in heterosexuals. HIV is a systemic immunosuppressive disorder which causes a depletion of CD4+ T cells and develops into the acquired immunodeficiency syndrome - AIDS. Africa is the continent most affected by HIV/AIDS with the southern parts of Africa having the highest prevalence rates compared to the rest of Africa. Statistics indicate that AIDS is responsible for 3% of deaths in children worldwide - one in seven people dying of an HIV-related illness is a child under the age of 15 years. It was stated by the WHO that countries should develop improved antiretrovirals regimes for the prevention of mother-to-child transmission. Difficulties in administering antiretrovirals (ARVs) to patients (especially children) are the strict dosage regimes and the severe adverse reactions. These factors complicate patient adherence. The list of problems in treating patients is endless and includes the distribution, stability as well as the low efficacy of these drugs. Most of the above mentioned problems and obstacles related to ARVs and ARV treatment could be minimized or eliminated by the use of a stable and effective drug delivery system. Enhancing ARV treatment may be accomplished by the use of the Pheroid™ drug delivery system. Pheroids™ consists mainly of fatty acids and sterile nitrous oxide gassed water. Pharmacological active substances are entrapped into submicron and micron sized structures called Pheroids™. Research showed promising results and advantages in delivering drugs through oral and transdermal routes using Pheroid™ technology. The focus of this study was to test the possible enhancement of the efficacy of antiretrovirals using Pheroid™ technology. The assays used to study this possible enhancement were a modified neutral red and a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. These assays confirmed and illustrated the toxic and protective properties of the tested ARVs (stavudine, lamivudine and nevirapine). An MT-2 cell line was used and infected with an HIV-1 strain, SW7-TCL. Applying Pheroid™ technology in these assays resulted in massive cell death, due to increased ARV toxic levels within the cells. Viability tests proved that Pheroids™ had no effect on the viability of cells at the concentration typically used. This confirmed the enhancing properties of Pheroids™ in the delivery of drugs into the cells. The MTT assay was further adapted from a seven day incubation period to a three day incubation period. By using a low concentration series and a three day incubation period the loss of cells through toxicity was partially overcome. One of the problems that arose form this study was the non-reproducibility of the results. Absorbance levels fluctuated at specific concentrations of the same ARV, which cause difficulties in comparing results. This result was repeatedly confirmed in this syncytium forming infection model. In conclusion, Pheroid™ technology enhanced the delivery of ARVs into the cells although it resulted in cell death. Both the neutral red and MTT assays were found to be inaccurate but further development, research and assay optimization could result in improved in vitro studies. The article format was used for this thesis, as described in the general academic rules in section A.13.7.3 of the North West University. Chapter 1 deals with HIV/AIDS related problems, statistics and treatment obstacles. Chapter 2 is a summary of the cell viability assays used in this study. Pheroid™ technology and its application to ARV treatment are dealt with in chapter 3. The proposed article for submission in the journal Cell Death and Differentiation has been included in chapter 4. Some of the results from the study are reported in the article and annexures, whilst other results are shown and discussed in Chapter 5. Chapter 6 gives a conclusion and final summary of this study. All other experimental methods and results are enclosed in the annexures, as is the "Guide for authors" for the article. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Antiretroviral drugs have revolutionized HIV care and enabled better management of the infection thus allowing patients survive for many years. One proposed approach to increase access to such drugs in sub-Saharan Africa is to use of a reduced-dose alternative of the drug efavirenz, with 400 mg rather than regular 600 mg dose. This effectively would provide medication for 50 percent more persons with the same amount of active ingredient. However, antiretroviral drugs require high patient adherence to achieve intended therapeutic effect, and it is unclear if a reduced-dose therapy would have sufficient efficacy, and if it would lead to an increased risk of viral resistance. The time profile of drug plasma concentration and corresponding long-term viral load was estimated using integrated population PK/PD simulations, with model parameters based on selected research studies. The results suggest a reduced dose 400 mg, rather than 600 mg regular dose, efavirenz in HIV therapy would place strict demands on patients to maintain very high adherence levels, at least 80-90 percent, to maintain sufficient drug concentration in blood plasma, and to minimize risk of viral failure. However, it is relatively rare for HIV therapy programs in sub-Saharan Africa to consistently achieve such high adherence levels. In addition, if patients are co-administered rifampin, a drug widely used in TB care, this increases hepatic metabolism and plasma clearance rate, resulting in further reduced average drug plasma concentration. These findings suggest a reduced dose efavirenz treatment alternative may be most (only) relevant for patient categories expected to maintain high adherence; and in particular among persons who have been confirmed to have CYP2B6 genotype consistent with inherently lower drug metabolism. At usual adherence levels it is estimated a reduced dose alternative may increase the share of patients at risk of viral failure by 5 to 15 percent vs. regular dose of 600 mg.

Les pays africains au sud du Sahara ont vu leur nombre de patients sous traitement antirétroviral (ARV) croître de façon rapide depuis 2005. Si l’impact individuel et collectif de cette montée en puissance des traitements est positif dans l’ensemble, les défis demeurent nombreux en termes de dépistage, d’observance, d’adhésion aux soins, de résistance aux ARV, de dépendance vis-à-vis des bailleurs de fonds, et de disponibilité des personnels. Dans ce contexte, la question du moment idéal pour proposer le début du traitement ARV doit être abordée de façon médicale individuelle (quel est le rapport bénéfices/risques individuel à débuter à des seuils différents ?), mais également de façon collective en terme de bénéfices et risques pour la communauté, d’organisation des soins, d’analyse médico-économique, de prioritisation et d’équité. Cette thèse, qui est une thèse de recherche clinique, aborde le premier volet de la question, celui des bénéfices et des risques pour l’individu à débuter un traitement plus tôt. Sur ce sujet, le raisonnement a beaucoup évolué au cours des 15 dernières années. Après l’arrivée des multithérapies ARV à la fin des années 1990, la crainte de la toxicité des médicaments a d’abord incité à une approche prudente, et à recommander le seuil de début à 200 CD4/mm3 chez les personnes asymptomatiques. Cette crainte de la toxicité a conduit au début des années 2000 à essayer de pratiquer des « interruptions programmées » d’ARV, pour tenter d’obtenir le maintien au dessus d’un seuil de 200 CD4/mm3, tout en limitant l’exposition aux médicaments. Nous avons participé à un de ces essais d’interruptions programmées en Côte d’Ivoire, au cours duquel nous avons contribué à affiner les connaissances sur la toxicité des ARV (Moh, Antivir Ther 2005). Les essais d’interruptions programmées ont conduit à constater que : (i) les personnes qui interrompaient entre 350 et 250 CD4/mm3 avaient plus de risque de morbidité sévère que celles qui n’interrompaient pas, (ii) les personnes qui débutaient leur premier traitement avant 350 CD4/mm3 avaient moins de risque de morbidité que celles qui débutaient plus tard (Moh, AIDS 2007), et (iii) dans l’essai d’interruption Trivacan réalisé en Côte d’Ivoire, cette morbidité sévère intermédiaire était plus fréquente que dans l’essai SMART réalisé sur d’autres continents, et avait un spectre différent, dominé par la tuberculose et les maladies bactériennes sévères. Les conclusions de ces essais ont donc été que le traitement ARV devait être débuté beaucoup plus tôt que ce qui était auparavant recommandé, et que ceci était probablement encore plus vrai en Afrique sub-Saharienne que dans le reste du monde. En 2008, nous avons lancé en Côte d’Ivoire l’essai Temprano ANRS 12136, dont l’objectif est d’évaluer les bénéfices et risques d’un traitement ARV précoce avec ou sans 6 mois de prophylaxie par isoniazide (INH) chez des adultes infectés par le VIH-1 ayant entre 250 et 800 CD4/mm3. De Mars 2008 à Juillet 2012, 2076 adultes ont été inclus dans l’essai Temprano, dont le suivi se terminera en décembre 2014. L’état du suivi est bon, et les incidences de morbidité et mortalité actuellement constatées sont conformes aux hypothèses du protocole. La pratique de la prophylaxie par INH s’avère bien tolérée, et la procédure choisie par notre équipe (radiographie de thorax systématique et période tampon d’observation de un mois avant le début de l’INH) apporte une grande sécurité de prescription (Moh, Plos One, manuscrit en révision). Notre équipe a traversé une crise politico-militaire au 1er semestre 2011, qui n’a pas eu de retentissement sur la qualité de l’essai en cours. Cette crise a par contre eu des effets délétères pour les patients sous traitement ARV, puisque les échecs virologiques retardés sont significativement associés au fait d’avoir été sous traitement pendant cette période (Moh, manuscrit soumis). . / The African countries situated in the South of the Sahara have seen their number of patients under antiretroviral therapy (ART) grow rapidly since 2005. If the individual and collective impact of this rise of the treatments is positive overall, challenges remain in terms of screening, compliance, accession to care, resistance to ARTs, dependence on donors, and availability of the staff. In this context, the question of the ideal time to propose initiation of ART must be addressed in the individually medical way (what is the individual benefit-harm ratio to start at different thresholds?) but also collectively in terms of benefits and risks for the community, organization of care, medico-economic analysis, prioritization and equity. This thesis, which is a clinical research thesis, addresses the first part of the question, the benefits and risks for the individual to start treatment earlier. On this subject, the rationale has changed considerably over the past 15 years. After the arrival of ART multitherapy at the end of the 1990s, the fear of drug toxicity first prompted a cautious approach, and to recommend the threshold from beginning to 200 CD4/mm3 in the asymptomatic people. This fear of toxicity led in the early 2000s to try to practice "scheduled interruptions" of ARTs, to try to get the maintenance above a threshold of 200 CD4/mm3, in limiting exposure to the drug. We have participated in one of these trials of interruptions programmed in Côte d'Ivoire, in which we have helped to refine the knowledge on the toxicity of ARTs (Moh, Antivir Ther 2005). Testing scheduled interruptions led to see that: (i) persons who interrupted between 350 and 250 CD4/mm3 had greater risk of severe diseases than those who didn’t interrupt, (ii) persons who started their first treatment prior to 350 CD4/mm3 had less risk of morbidity than those who started later (Moh, 2007 AIDS), and (iii) in trial interruption Trivacan launched in Côte d'Ivoire, this intermediate severe morbidity was more frequent than in the SMART trial carried out on other continents, and had a different spectrum dominated by tuberculosis and severe bacterial diseases. The findings of these trials were that the ART should be started much earlier than was previously recommended, and that this was probably even truer in sub-Saharan Africa than in the rest of the world. In 2008, we launched in Ivory Coast the clinical trial, Temprano ANRS 12136, whose objective is to assess the benefits and risks of early ART with or without 6 months of prophylactic isoniazid (INH) in HIV-1 infected adults with CD4 250 and 800/mm3. From March 2008 to July 2012, 2076 adults were included in the trial Temprano, which follow-up will be completed by December 2014. The state of the follow-up is good, and the impact of morbidity and mortality currently observed are consistent with the assumptions of the Protocol. The practice of INH prophylaxis is well tolerated, and the procedure chosen by our team (systematic chest x-ray and period buffer observation of one month before the beginning of the INH) brings a prescription safety (Moh, Plos One manuscript in review). Our team went through a crisis politico-military 1St half 2011, which had no impact on the quality of the ongoing trial. This crisis has however had deleterious effects for patients under ART, since delayed virological failure are significantly related to the fact of having been under treatment during this period (Moh, submitted manuscript).

L'utilisation des antirétroviraux (ART) a permis une augmentation de la durée des patients infectés par le VIH. Par ailleurs, les comorbidités, retrouvées au cours du vieillissement physiologique, semblent être plus fréquentes et d'apparition plus précoce ce qui pourrait suggérer une modification du programme de vieillissement chez ces patients. L'étude ANRS EP45 « Aging » (clinicalTrials.gov, NCT01038999) a pour objectif d'analyser chez des patients infectés par le VIH traités ou non les mécanismes cellulaires connus pour être impliqués dans le vieillissement. Les PBMC d'une cohorte de 130 patients infectés par le VIH 1 appariés en âge et en sexe avec 49 sujets séronégatifs ont été analysés. Trois centres spécialisés (Marseille, Montpellier, Nice) ont recruté des patients infectés naïfs ou sous première ligne de traitement. Les résultats présentés dans ce manuscrit rapportent l'analyse des mitochondries et des lamines nucléaires. La maturation de la lamine A ne semble pas modifiée dans les PBMC de patients sous traitement contenant un inhibiteur de protéase. Cependant, ces cellules pourraient ne pas être le modèle le plus adapté pour explorer ce volet. D'autre part, l'infection est responsable d'anomalies mitochondriales dans les lymphocytes, partiellement corrigées par les traitements antirétroviraux qui modifient les mitochondries des monocytes moins sensibles à l'infection. Bien que les secondes générations de ART soient moins toxiques que les premières, leurs effets secondaires pourraient néanmoins, sur « le long terme » et/ou généralisés à l'ensemble de l'organisme, être l'un des facteurs modifiant le programme de vieillissement de ces patients. / Antiretroviral therapy (ART) has increased life expectancy in HIV-infected patients. Moreover, some age-related disorders were found to be more frequent in HIV infected and treated patients than in an age-matched general population, suggesting a modified time course of aging in HIV infected patients. The ANRS EP45 « Aging » study (clinicalTrials.gov, NCT01038999) investigated in PBMC from HIV-1 infected patients under treatment or not the cellular mechanisms known to be involved in aging. The study was performed on a cohort of 130 patients HIV-1 infected age- and sex-matched with 49 seronegative control subjects. Patients never treated with ART (naïve) or under first line were recruited by 3 AIDS centres (Marseille, Montpellier, Nice). Results presented here describe explorations of mitochondria and nuclear lamin. No alteration of lamin A maturation was detected in PBMC from HIV-1 infected patients under treatment with protease inhibitor. However, these cells could not be the most appropriate models to investigate lamin A-related aging pathway. On another hand, mitochondrial modifications were observed in lymphocytes from HIV infected naive patients. These alterations were only partly rescued by ART whereas its induced slight changes in monocytes that appeared to be less sensitive to infection. While second generation of ART are less toxic than the first one, their secondary effects, due to long term exposure and/or generalised to different tissues, could lead to a modified time course of aging in HIV infected patients.

M.Sc. (Chemistry) / Human immunodeficiency virus (HIV) is continuously rewriting medical history as one of the diseases affecting humankind. Current treatments available for HIV, namely antiretrovirals (ARVs), do not completely eradicate the virus from the body, leading to life time commitment. Many ARVs suffer from high toxicities and unpleasant side effects; as a result many patients do not adhere to the treatment. Nanoparticles (NPs) used as drug delivery systems (DDS) hold tremendous potential, since they can easily protect the drug from external environment and enter the human cells to deliver drugs. Therefore, the main objective of this work was to load two ARVs, namely lamivudine (LAM) and efavirenz (EFV), into a biodegradable, biocompatible poly(epsilon-caprolactone) (PCL) polymer based NPs. LAM is a hydrophilic drug suffering from low half life of 5 to 7 hours and many unpleasant side effects. EFV is a hydrophobic drug suffering from low aqueous solubility (4 μg/ml), which leads to a limited oral absorption and low bioavailability (40-45%).

Magister Public Health - MPH / Background: Antiretroviral therapy (ART) was rolled out in South Africa in the public sector in 2004 and the treatment coverage has increased over the years to 56% in 2016. The increased treatment coverage has the potential to increase the level of HIV drug resistance. Drug resistance presents a major challenge to the management of HIV infection through antiretroviral therapy at the population level. The aim of this study was to determine the impact of the public sector antiretroviral therapy rollout on the prevalence of HIV drug resistance in South Africa and the factors associated with drug resistance. Methodology: A cross-sectional analytical study was used to determine the prevalence of drug resistance before and after ART rollout. The study population was HIV infected South Africans (infected between 1996 and 2011) who were not on antiretroviral therapy. The study sample was therapy naïve HIV infected South Africans who participated in published studies conducted between 1996 and 2011. HIV DNA sequences and associated data (participants’ age, gender, geographic location and estimated year of HIV infection) were accessed through the Los Alamos HIV Database. The database contains all HIV DNA sequences and associated data from all published studies and the data was freely accessible. A descriptive analysis was carried out on the data to determine characteristics of the study sample. Drug resistance mutations were detected using Calibrated Population Resistance Program on the Stanford University HIV Drug Resistance database. The output from the Calibrated Population Resistance Program analysis were used to determine the prevalence of drug resistance mutations. Results: There were 1701 DNA sequences obtained from the Los Alamos HIV Database for the three gene regions targeted by ART (reverse transcriptase, protease and integrase). Of these, 604 (35,5%) were for reverse transcriptase, 794 (46,7%) were for protease and 303 (17,8%) were for integrase. There was overrepresentation of DNA sequences from female participants (91%). There was no significant difference in the prevalence of drug resistance mutations between 1996-2004 (before ART rollout) and 2005-2011 (after ART rollout) in all the drug classes. There was also no association between drug resistance and age as well as gender. Conclusion: The data from this study suggest that the public sector rollout of ART did not result in an increase in the prevalence of drug resistance mutations in therapy naïve HIVinfected South Africans. There is need for further studies, which have a wider coverage of the South African population.