Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2006. / Acquired immunodeficiency syndrome (AIDS) was first reported in 5 homosexual men in Unite States of America in 1981 as a series of opportunistic infections which occasionally occurred in adults. Subsequently, it has been achieved that human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS and this aetiological agent has spread all over the world. The virus primarily attacks CD4+ T cells and gradually leads to progressive depletion of CD4 T lymphocytes from peripheral blood and lymphoid organs. Since CD4+ T cells are vital immune cells in induction and regulation of both cell-mediated and humoral immune responses, depletion of these cells ultimately results in a profound immunodeficiency characterized by susceptibility to variety of opportunistic infection. Apoptosis have been commonly proposed as the mechanism of CD4 depletion because elevated levels of apoptosis were observed in HIV-1 infected individuals (Ameisen et al., 1991; Groux et al., 1992 & Oyaizu et al., 1993). Nevertheless, there was evidence showing that HIV-1 infected cells died not from apoptosis (Bolton et al., 2002) and another study reported that inhibition of apoptosis resulted in high viral production (Antoni et al., 1995). These controversial views indicated that the mechanism of CD4 depletion and the immuno-pathogenesis of apoptosis should be considered. As a pilot sub-study, eight HIV-1 infected subjects were enrolled to determine the methods in measuring apoptosis. Three different cell separations: (1) whole blood cells, (2) buffy coat cells and (3) isolated PBMCs were prepared to determine whether different cell preparations result in different measurements of apoptosis. In addition, FITC-labelled Annexin V, an early marker of apoptosis, and flow-cytometer based scatter methods based on characteristics of apoptotic cells were used to investigate the difference in analytical methods in determining the levels of apoptosis. Firstly, it was found that whole blood samples yielded more precise measurements in measuring apoptosis, followed by Buffy coat and then PBMC samples. Secondly, this sub-study also indicated that the scatter method as well as fluorenscent labelled Annexin V could be useful markers for apoptosis. Secondly, different surface markers of apoptosis were used to investigate apoptosis in HIV-1 infected adults. Fifty-eight HIV-1 infected adults were involved in this sub-study. They were classified into three categories based on CDC CD4 category classification (CDC, 1993). According to the data, the level of apoptotic CD4+ T cells measured by the scatter method was high in CD4 category 1, decreased in category 2 and finally increased again in category 3. This tendency was in parallel with CD95 (Fas) expression on CD4+ T cells. The curve formed a “V” shape according to the three CD4 categories. Together with the gradually increased plasma viral load, these data reflect an activated immune response at early stage of infection and under controlled viraemia. This possibly represents the immune response trying to eliminate infected cells as a means of survival. The high level of apoptosis in category 3 could indicate a disordered immune system accounting for the rapid loss of CD4+ T cells and progression to AIDS. A novel finding of this study was the presence of two CD4+ populations in 10 HIV-1 infected subjects, which were CD4dim and CD4bright. These 10 subjects had relatively high CD4 count and low viral replication. Statistical analysis showed they had significantly higher levels of apoptosis in CD4 and CD8 T lymphocytes, measured by the scatter method, than those subjects presenting single CD4 population. In addition, when comparing the two CD4 subpopulations, it was found that CD4dim cells had significant higher level of apoptosis and CD95 expression than the CD4bright cells. Finally, the virological and immunological effects of antiretroviral therapy (ART) were investigated in two cohorts of HIV-1 infected children. Fourteen HIV-1 infected children were involved in investigation of 12-month long-term effect, while another five children were involved in a short-term 1-month follow-up study. In addition, a different assay of detecting apoptosis: terminal deoxynucleotidyltransferase deoxyuridine triphosphates nick end labeling (TUNEL) was conducted to measure the level of apoptotic PBMCs. According to the findings from 12-month and 1-month sub-studies, it appeared that ART could be effective in suppression of viral replication at an early stage. However, the immunological effect, such as CD4 reconstitution, could only be seen as a long-term effect, since immune recovery would take a long time. In addition, different regimens containing protease inhibitors (PIs) might be more effective in inhibiting apoptosis than non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: S. aureus causes serious infections in the hospital and community settings. The rate of MRSA infections are rapidly increasing worldwide. Currently, at Tygerberg hospital, approximately a third of S. aureus isolates are MRSA. This was the first epidemiological study of S. aureus conducted at Tygerberg Hospital that included prospective clinical data on patients with S. aureus bacteraemia together with spa typing of strains and the detection of the mecA and pvl genes in a multiplex PCR. Clonal cluster groups of S. aureus isolates were obtained by BURP analysis and compared to international important clones. The molecular epidemiology of hospital acquired (HA), health-care associated (HCA) and community acquired (CA) S. aureus bacteraemic strains at this hospital was examined. Lastly, repeat isolates of patients were collected to analyse any possible organism-related factors associated with persistent and recurrent bacteraemia. We investigated a total number of 113 S. aureus strains from 104 patients (70% MSSA, 30% MRSA). Repeat strains consisted of nine isolates (from 5 patients). All isolates were obtained from blood cultures collected during the period March 2008 to May 2009. Phenotypic and genotypic detection of methicillin resistance correlated well. According to the literature, most CA-MRSA strains are distinguishable from HA-MRSA strains based upon the presence of the PVL toxin. However, no CA-MRSA was detected in our study, therefore the association between HA-MRSA versus CA-MRSA strains could not be analysed. In this study, CA-MSSA was identified in 22% of all MSSA isolates versus 0% CA-MRSA. PVL positive strains were found in 22.7% of all MSSA isolates with no detection in MRSA isolates. It was noted that MRSA strains clustered in spa CC-701 and CC-012, whereas CC-002 only contained MSSA strains. Likewise HA-strains representing the majority of MRSA strains also clustered in spa CC-701 and CC-012. Forty nine spa types were identified in 89.3% of all isolates, whereas 9.7% of these strains were non-typeable. Five novel spa types were revealed. We detected a diverse number of spa-types that correlated to international clones. The most predominant spa type found in our setting was t037 (only in MRSA), followed by t891. According to the literature, t037 is associated to the Brazilian/Hungarian clone (SCCmec type III; ST 239). Our findings, as well as other South African studies, indicate that t037 has been identified in clinical strains from numerous provinces in South Africa. Interestingly, all isolates from spa type t891 were PVL positive MSSA. Bacteraemia cases were predominantly related to catheter sepsis, followed by skin and soft tissue infections (SSTI). Only one persistent bacteraemia case was identified related to a HA-SSTI. Recurrent bacteraemia cases were found in patients on dialysis for chronic renal failure and in burns patients related to intravascular catheter infections. The local epidemiology of S. aureus and the prevalence rate of different strains are important to investigate. The information provided contributes to the epidemiology of staphylococcal strains causing bacteraemia in our setting. These insights are useful for optimal diagnostic and therapeutic measures. The techniques developed can be used to identify outbreaks and recurrent infections. / AFRIKAANSE OPSOMMING: S. aureus veroorsaak ernstige infeksies in die hospitaalomgewing en in die gemeenskap. Wêreldwyd, neem metisillien-weerstandige S. aureus (MRSA) infeksies vinnig toe. Huidiglik by Tygerberg hospitaal is ongeveer ‘n derde van S. aureus isolate MRSA. Hierdie is die eerste epidemiologiese studie by Tygerberg hospitaal wat prospektiewe kliniese data van pasiënte met S. aureus bakteremie saam met spa tipering en aantoning van die mecA en pvl gene in ‘n multipleks PKR insluit. Klonale groepe (spa-CC) van MRSA en MSSA isolate is deur BURP analise verkry, en vergelyk met internasionaal belangrike klone. Die molekulêre epidemiologie van hospitaalverworwe (HA), gesondheidsorgverworwe (HCA) en gemeenskapsverworwe (CA) S. aureus bakteremie by hierdie hospitaal is ondersoek. Laastens, oorspronklike en daaropvolgende herhaal isolate is gekollekteer om moontlike organisme- faktore geassosieerd met persisterende en herhalende bakteremiese episodes te analiseer. Ons het in totaal 113 S. aureus isolate van 104 pasiënte ondersoek (70% MSSA, 30% MRSA). Nege isolate (van 5 pasiënte) was herhaal isolate. Alle isolate was afkomstig vanaf bloedkulture wat gedurende die periode Maart 2008 tot Mei 2009 gekollekteer is. Fenotipiese en genotipiese aantoning van metisillien weerstandigheid het goed gekorreleer. Volgens die literatuur kan die meeste CA-MRSA isolate van HA-MRSA isolate onderskei word op grond van die teenwoordigheid van die PVL toksien. Geen CA-MRSA is egter in ons studie gevind nie, dus kon die assosiasie tussen HA-MRSA en CA-MRSA isolate nie ondersoek word nie. CA-MSSA was in 22% van alle MSSA geidentifiseer teenoor 0% CA-MRSA. PVL is in MSSA isolate gevind (22.7% van alle MSSA) maar glad nie in MRSA nie. Dit is opgemerk dat MRSA isolate hoofsaaklik in spa CC 701 en CC-012 kloongroepe voorkom, teenoor kloongroep CC-002 wat slegs MSSA isolate bevat het. Soortgelyk het HA-isolate wat die meerderheid van MRSA isolate verteenwoordig het ook in kloongroepe 1 & 2 gegroepeer. Nege-en-veertig spa tipes is geïdentifiseer in 89.3% of alle isolate en 9.7% was nie-tipeerbaar. Vyf nuwe spa tipes is getoon. Ons het ‘n diverse aantal spa-tipes geïdentifiseer wat met internasionale klone gekorreleer het. Die mees dominante spa tipe in ons omgewing was t037 (slegs in MRSA), gevolg deur t891. Volgens die literatuur word t037 met die Brasiliaanse/Hongaarse kloon geassosieer (SCCmec tipe III; ST 239). Ons bevindings, asook ander Suid Afrikaanse studies, dui aan dat t037 in kliniese isolate vanaf talle provinsies in Suid-Afrika aangetoon is. Van belang is dat al die isolate van spa tipe t891 MSSA en PVL positief was. Bakteremiese gevalle was hoofsaaklik geassosieer met kateter-sepsis, gevolg deur vel en sagteweefsel infeksies (SSTI). Slegs een persisterende bakteremiese geval was geïdentifiseer geassosieer met HA-SSTI. Herhalende bakteremiese episodes is in pasiënte op dialise vir kroniese nierversaking en in brandwonde pasiënte met intra-vaskulêre kateter infeksies aangetoon. Die lokale epidemiologie van S. aureus en die prevalensie koers van verskillende stamme is van belang. Hierdie inligting dra by tot kennis van die epidemiologie van stafilokokkale stamme wat in ons omgewing bakteremie veroorsaak. Hierdie insigte is nuttig vir optimale diagnostiese en terapeutiese riglyne. Die tegnieke wat ontwikkel is, kan gebruik word om uitbrake en herhalende infeksies te identifiseer.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / Pneumocystis jirovecii is a significant contributor to the burden of disease in immunocompromised patients. The polymerase chain reaction (PCR) is more sensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough and treatment failures have been reported, and associated with mutations at codons 55 and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic or population genetic models have been successful in elucidating P. jirovecii intraspecies strain relatedness. Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescence microscopy (IF); 2) Determine the extent of co-infecting pathogens associated with Pneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNA strain types, and model lineage evolution employing a coalescent-theory based statistical parsimony network analysis; 4) Investigate the possible emergence of cotrimoxazole-resistant strains Methods: PCR was evaluated on clinical specimens employing: ITS nested; DHPS single and nested; DHFR nested; major surface glycoprotein (MSG) heminested; mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetube nested, and real-time 5S rRNA PCR. Retrospective analysis of co-infecting pathogens seen in PcP patients was conducted. ITS regions were amplified, cloned and sequenced. Statistical parsimony was applied for coalescence based network genotype analysis. DHPS genome walking was attempted and DHPS and DHFR primer annealing sites explored. Amplified DHPS and DHFR genes were cloned and sequenced. Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtime PCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review of clinical records suggested a high rate of false-positive IF results. P. jirovecii was detected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositive HIV-negative patients. P. jirovecii was detected in 45% HIV-negative patients. The most prevalent ITS type was Eg. Four new combinations: Eo, Je, Ge, No; 11 new ITS1 and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and designated u. More than one strain type was detected in 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2 nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currently employed primers amplified DHPS and DHFR genes from 53 and 27 specimens, respectively. Newly designed DHPS primers increased detection in 3 / 28 previously DHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified and sequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFR Ala67Val was detected in three specimens and a new DHFR genotype (Arg59Gly; C278T) was demonstrated. Conclusions: The study emphasises the need to evaluate PCR primers against local strains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF and discordant results resolved with clinical evaluation. Co-infection with P. jirovecii and M. tuberculosis occurs in South Africa, and treatment for both pathogens is recommended when demonstrated by the laboratory. ITS genotyping employing statistical parsimony network analysis suggests type Eg as major ancestral haplotype, and supports recombination contributing to strain diversity worldwide. DHPS mutations may signal emergence of resistance to cotrimoxazole in South Africa, however, low sensitivity of primers limits surveillance efforts.

p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.

Conventional drug susceptibility testing techniques, the ‘gold standard’ for M. tuberculosis are slow, requiring about 3-6 weeks from a positive culture. This diagnostic delay, before initiation of appropriate treatment, contributes to increased transmission rates. Molecular techniques provide rapid results and therefore present an alternative to conventional tests. The aim of this project was to develop an inhouse reverse line blot hybridization assay (RIFO assay) that could detect mutations associated with Rifampicin resistance directly in clinical specimens of patients in KwaZulu Natal. A 437 bp region of the rpoB gene was sequenced to ascertain the most frequently occurring mutations conferring resistance to rifampicin in isolates in KwaZulu-Natal. Wildtype and mutant probes designed to target these mutations, were immobilized on a Biodyne C membrane. Hybridization conditions were optimized using biotin labeled PCR products from culture. Detection was performed with peroxidase labeled streptavidin using enhanced chemiluminescence. Four DNA extraction methods were evaluated on sputum specimens to determine the one with the least inhibitory effect on amplification. A total of 11 mutations were found in 236 clinical isolates: 531TTG (109, 58.3%), 516GTC (26, 13%), 533CCG/516GGC (20, 10%), 533CCG (18, 9.6%), other mutations < 5% each. The chelex extraction method was found to be optimal for removing inhibitors in sputum specimens. Sputum specimens of 404 patients hospitalized at King George V Hospital between 2005 and 2006 were rifoligotyped. The RIFO assay was optimised on clinical isolates and then applied to sputum specimens. The RIFO assay on culture and sputum correlated well with the DST (sensitivity 92% and 94% respectively). However, the specificity was very low in both culture and sputum specimens compared to DST (38% and 35% respectively). This could be attributed to the presence of silent mutations, mixed infections, mixed populations of bacteria or the small number of susceptible strains used in this study. The in-house RIFO assay can be used directly on sputum specimens to predict Rifampicin resistance and therefore MDR-TB in less than a week compared to the gold standards. A total of 43 samples can be tested simultaneously at low cost and the membrane is reusable compared to commercial kits such as the Hains test that is expensive and strips are not reusable. A similar assay can be designed to target mutations for the detection of XDR-TB. Future studies should be conducted in a clinical setting on patients with sensitive strains to increase the specificity. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Squamous cell carcinoma of the oesophagus (OSCC) is a common malignancy that occurs with high frequency in certain parts of the world, including South Africa. The aetiology of OSCC has remained unclear although many studies suggest that it is caused by a combination of variable risk factors. Recent reports implicate a variety of genetic factors in the carcinogenesis of OSCC but their involvement is yet to be defined. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2010.

The increasing frequency of enterococci as a major cause of nosocomial infections and the transmission of these organisms amongst hospital patients demands a greater awareness of the Enterococcus. Therapy of enterococcal infections is complicated by the pathogens continually changing resistance patterns to many broad-spectrum antibiotics. In addition, the ability of enterococci to cause serious invasive infections including endocarditis and septicaemia with associated high mortality rates; prompted this study which was aimed at identifying the biological properties of enterococci isolated from blood cultures of patients admitted at King Edward VIII hospital, Durban. Enterococci were identified to species level by the API 20 Strep system which identified 68% and a conventional biochemical system of Facklam and Collins which identified 100% of the isolates.The emergence of beta-Iactamase producing enterococci in other countries encouraged the testing of all isolates for this enzyme. All were beta-Iactamase negative. The reported false susceptibility for aminoglycosides and cephalosporins with blood enriched media encouraged the testing of these antibiotics with and without the supplementation of 5% lysed blood. The results showed that an average false susceptibility of 55 % occurred for gentamicin and 35% for tobramycin and netilmicin. The cephalosporins affected, cefotaxime and cefuroxime showed a false susceptibility of 28% and 17% respectively. The choice of treatment for serious enterococcal infections is a syllergistic combination of a beta-Iactam antibiotic plus an aminoglycoside for enterococci with intrinsic low-level resistance. The development of high-level aminoglycoside resistance, MIC 22000,ug/ml results in loss of synergism. This study showed that 26.4 % of enterococcal isolates displayed high level aminoglycoside resistance i.e. to gentamicin and streptomycin. Time-kill study showed reduced killing rate for these organisms for the beta-Iactams and glycopeptides with low-level gentamicin resistance. The results confirmed that a cell-wall active agent combined with gentamicin can be successfully used for enterococcal therapy if the organism has intrinsic low-level resistance to this amino glycoside. Pulsed-field gel electrophoresis (PFGE) carried out on a selected number of Enterococcus faecalis and Enterococcus faecium with high-level aminoglycoside resistance showed a variability in the restriction endonucelase digestion patterns. This suggests independent development of high-level gentamicin resistance and not clonal expression. The ease and reliability with which enterococcal isolates may be typed using this technique to compare different strains represent a significant advance. / Thesis (M.Med.Sc.)-University of Natal, 1999.

Introduction APOBEC3G and other related cellular cytosine deaminase family members have potent antiviral activity. In the absence of HIV-1 Vif, APOBEC3G mutates the viral DNA during viral reverse transcription. Our knowledge of the Vif-APOBEC3G interaction in human populations infected with subtype C HIV-1 is limited. Investigation of interactions between HIV and its host is crucial as it can ultimately be exploited in vaccine and therapy design. We hypothesised that certain APOBEC3G haplotypes and/or their expression in peripheral blood mononuclear cells of seroconverters affect viral setpoint and CD4+ T cell counts. We also hypothesised that certain APOBEC3G genetic variants are associated with increased frequency of G to A hypermutations during primary HIV-1 infection and that Vif variability influences disease progression and its ability to neutralise APOBEC3G haplotypes. Methods Our South African study cohort consisted of females at high risk for HIV-1 infection and women with known recent HIV-1 infection. We used quantitative real-time PCR to measure APOBEC3G expression in HIV- and HIV+ samples during primary infection. APOBEC3G variants were identified by DNA sequencing and TaqMan Genotyping. The HIV-1env gene was sequenced to assess Env diversity and the extent of APOBEC3G induced hypermutations. Vif variability was assessed by plasma derived clonal Vif sequences (n= 10-20 per patient) and Vif function was assessed by APOBEC3G degradation assays and HIV-1 infectivity assays. Results We found no correlation between APOBEC3G expression levels and plasma viral loads (r=0.053, p=0.596) or CD4+ T cell counts (r=0.030, p=0.762) in 32 seroconverters. However, APOBEC3G expression levels were significantly higher in HIV- individuals compared to HIV+ individuals (p<0.0001) including matched pre- and post-infection samples from the same individuals (n=13, p<0.0001). Twenty five single nucleotide polymorphisms (SNPs) were identified within the APOBEC3G region. SNP 186R/R was associated with significantly higher viral loads (p=0.0097) and decreased CD4+ T cell levels (p=0.0081), indicating that 186R/R has a negative effect on HIV restriction. Overall HIV-1 env sequences contained a higher number of APOBEC3F compared to APOBEC3G-induced hypermutations and the number of APOBEC3F-induced hypermutations correlated negatively with viral load (r= -0.6, p=0.006) and positively with CD4 T cell counts (r=0.6, p=0.004). We cloned and sequenced a total of 392 subtype C Vifs, which showed an interpatient diversity of 6.2% to 19.2% at the amino acid level. Interestingly, Vif sequence comparison showed a strong preference for a Lysine or a Serine at position 36 for APOBEC3G 186R/R and APOBEC3G 186H/H individuals, respectively. Selected natural subtype C Vif alleles had greater ability to counteract wild type APOBEC3G 186H as compared to the APOBEC3G 186R variant as shown by both functional and HIV infectivity assays. Conclusions In conclusion, APOBEC3G expression in peripheral blood mononuclear cells does not correlate with viral loads or CD4+ T cell counts during primary HIV-1 subtype C infection. However, genetic variants of APOBEC3G may affect HIV-1 pathogenesis. Amino acid changes in Vif may influence its anti-APOBEC3 activity. HIV-1 subtype C Vifs may have adapted to counteract the more active wild type APOBEC3G as compared to the less active APOBEC3G 186R variant. These studies have improved our understanding of viral-host interactions in African populations and HIV-1 subtype C infections. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2014.

The management of HIV-associated tuberculosis (TB) is complicated by the pharmacokinetic interactions between rifampicin (RMP) and co-administered protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors. Rifabutin (RBT) is an alternative rifamycin, preferred in patients requiring PIs. Recent studies suggest the current recommended dose of RBT in combination with boosted lopinavir (LPV/r) is suboptimal and there are insufficient pharmacokinetic data evaluating the interaction between RBT coadministered with efavirenz (EFV) and nevirapine (NVP). Pharmacogenomic studies have shown that RMP concentrations are lower in patients from sub-Saharan Africa with polymorphisms of the SLCO1B1gene but there is currently no data on the pharmacogenetic determinants of RBT exposure. The pharmacokinetics of RBT were evaluated at two different doses in HIV co-infected patients before and after the introduction of LPV/r, EFV and NVPbased antiretroviral therapy (ART). After six weeks of standard TB therapy, RBT 300 mg daily was started for four weeks. Thereafter patients were randomized to receive either RBT 150 mg daily or RBT 150 mg three times a week (TPW) with LPV/r, RBT 300mg or 450mg with NVP or RBT- 450mg or 600mg with efavirenz. After four weeks on the first RBT dose, patients switched to the alternate dose and continued until the end of TB treatment. Serial RBT and 25-O-desacetylrifabutin (dRBT) concentrations were measured during a dose interval before patients switched RBT doses. The median AUC0-24 and Cmax, of RBT in patients taking 150mg RBT TPW was significantly reduced when compared to the other treatment arms. 86% of patients whilst on this intermittent RBT arm had an AUC0-24 < 4.5 μg.h/mL, level that has been associated with acquired rifamycin resistance. Rifabutin exposure was maintained within the range of AUCs that have been shown to prevent acquired rifamycin resistance (ARR) with 150mg daily dosing in combination with LPV/r. In addition, the combination of RBT with NVP 300mg resulted in significantly increased exposure of RBT, with significantly higher exposure observed with 600mg RBT. However, the combination of RBT 450mg with EFV resulted in RBT exposure lower than 300mg RBT given alone in the same patients, whereas RBT 600mg plus NVP results in bioavailability of RBT equivalent to 300mg given alone. Rifabutin was well tolerated at all doses. Only three grade 4 laboratory toxicities, elevated transaminases, neutropenia, and uveitis, possibly related to RBT were reported in patients taking NVP. SLCO1B1 rs4149032 C>T polymorphism occurs frequently in African patients in Durban and may be associated with low RBT bioavailability. These findings support recommendations for the higher dose of RBT in combination with LPV and EFV but not with NVP. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2012.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / The aim of this study was to investigate the anti-inflammatory and immunosuppressive effects of the synthetic progestins, MPA and NET-A on human cells in vitro. These injectable contraceptives are used extensively throughout the world, including Africa. The potential of these two synthetic hormones to have certain immunosuppressive and GC properties have previously been shown. Therefore, it was of concern to us to investigate whether these two hormones could possibly demonstrate any of these GC-like properties at contraceptive doses. This was achieved by determining the effects of these two synthetic hormones in vitro on certain immunologic parameters. Chapter 1 is a literature review on MPA, NET and GCs. This chapter starts with a short introduction that sets the scene. The mode of action, effectiveness, sideeffects as well as previously reported relevant data on both MPA and NET-A is portrayed in this review. Research on the known GC, Dex, is also included in the section dealing with GCs, because this synthetic hormone was used as a comparative GC in all our experiments. This chapter soon makes the reader realize how much evidence exists that indicate the possible immunosuppressive effects these two contraceptive hormones, in particular MPA, could have. The possible anti-inflammatory or pro-inflammatory effects of MPA and NET-A are investigated in Chapter 2. This was done in vitro by measuring the effects of these two synthetic hormones on the inflammatory markers, IL-6 and TNFα, by means of ELISA. In this chapter we demonstrate that MPA, even at contraceptive doses, exhibits significant anti-inflammatory properties on both cytokines tested, while NETA displayed considerably less anti-inflammatory tendencies. In its true antiinflammatory manner, we found that Dex significantly inhibited the release of both inflammatory markers from human monocytes. In Chapter 3, we investigated the effects of MPA and NET-A on the activation of human lymphocytes. This was achieved by flow cytometric measurement of the expression of the activation membrane marker CD69 by CD4 and CD8 T cells. Here we discovered that MPA had a very significant inhibitory effect on the activation of both CD4+ and CD8+ T cells, while NET-A only significantly inhibited the activation of CD8+ T cells. In addition, we found that the inhibition of CD4+ and CD8+ T cell activation by MPA was more or less the same as the known GC, Dex, and in some cases even more potent. Chapter 4 consists of an investigation of the effects of MPA and NET-A on the cytokines belonging to TH1 and TH2 subsets of CD4 T cells. This was achieved by determining whether MPA and/or NET-A targeted specific subsets of T helper cells by measuring the distinct regulatory cytokines, IFNγ and IL-4. The mechanism and role of the T helper subsets are discussed in the introduction of this chapter. Our results were portrayed as a ratio of TH2: TH1 on which the statistical analysis was done. In addition to the analysis done on the ratio, we analyzed the helper subsets separately in order to determine which subset(s) were influenced. The results of this chapter showed that neither MPA nor NET-A significantly affected either one of the helper subsets, while Dex significantly decreased this ratio. After our observed effects of MPA and NET-A on CD8 T cells, it became of interest in Chapter 5 to investigate the effects of these two synthetic hormones on the CD8 T cell-specific chemokine, RANTES. This was achieved by measuring the effects MPA and NET-A had on RANTES production in vitro by means of ELISA. Surprisingly, we discovered in this chapter that MPA and NET-A enhanced RANTES production before and after activation of CD8 T cells. We also found that Dex had the same effect on RANTES production, but to a lesser degree. Finally, a general conclusion depicting the significance and implications of our results as well as possible future research that is required is presented in Chapter 6. It was of great importance to discuss and interpret the magnitude of data generated out of all our experiments to the utmost of our capabilities. We found that MPA, even at contraceptive doses, displayed significant immunosuppressive as well as anti-inflammatory properties. NET-A, on the other hand, demonstrated weaker immunosuppressive properties in our research and no significant anti-inflammatory properties. These findings could have clinical implications in females being treated with these synthetic contraceptives. We also demonstrated significant variation found amongst genders in response to MPA, NET-A and Dex.