Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: HIV-positive patients have a significantly weakened immune system which makes them highly susceptible for opportunistic infections, requiring additional treatment. Cryptococcal meningitis and oropharyngeal candidiasis are treated with oral fluconazole. A great potential for drug-drug interactions (DDIs) between fluconazole and antiretrovirals (ARVs), efavirenz, nevirapine, and lopinavir/ritonavir, exists due to interference in common metabolic pathways. The outcome may result in the development of adverse drug reactions or drug resistance and treatment failure. Aim: The primary aim of this thesis was to evaluate the effect of fluconazole on the pharmacokinetics of efavirenz, nevirapine and lopinavir/ritonavir in HIV-infected patients diagnosed with cryptococcal meningitis or oropharyngeal candidiasis. Methods: A prospective study was conducted in 80 HIV-positive, treatment experienced adults (≥18 years old) treated in three different outpatient clinics in the Western Cape region. Patients were subdivided according to ARV regimen and the use of fluconazole. A sparse sampling design was used and corresponding ARV serum concentrations were determined by established HPLC and GC methods. Fluconazole serum concentrations were determined by a newly developed HPLC method. Patient characteristics, concomitant medications, clinical test data and ARV serum concentrations were included in a NONMEM generated, onecompartment, open pharmacometric model with first order elimination to detect any drugdrug interactions between fluconazole and the studied ARVs. The secondary outcome was to establish which patient characteristics influence ARV pharmacokinetics. Results: From 80 outpatients, a total of 276 ARV serum samples (137 efavirenz, 67 nevirapine and 72 lopinavir) were collected for pharmacokinetic evaluation. Efavirenz clearance was correlated with race and concomitant use of rifampicin. No significant covariates were established in the nevirapine model. In the lopinavir model, concomitant use of clotrimazole and the antituberculosis combination isoniazid, pyrazinamide and rifampicin were identified as significant covariates. Discussion: No significant effects of fluconazole on the pharmacokinetics of any of the studied ARVs were observed. Varying efavirenz plasma concentrations in different ethnic populations may be due to differences in gene expression particularly CYP2B6. Coloured patients had significantly lower efavirenz serum concentrations (56.8% decrease in clearance), which has not been previously described in the South African context. Although gender was not a significant covariate in the nevirapine model, female patients tended to have higher nevirapine serum concentrations. TB treatment in all patients receiving lopinavir consisted of a combination of isoniazid, pyrazinamide and rifampicin, each with different effects on CYP isoenzymes. The exact contributing factor of each drug in the ultimate decrease in lopinavir clearance (46.4%) can therefore not be established. Conclusions: Given the limitations of the sample size in the present study, no statistical significant effect of fluconazole on the pharmacokinetics of the investigated ARVs could be demonstrated. A retrospective analysis of the data showed various co-factors that influence the pharmacokinetics of the investigated ARVs. This data needs to be confirmed in a prospective study as the identified covariates are appropriate in the management of HIVinfected patients in the South African context. / AFRIKAANSE OPSOMMING: Agtergrond: HIV-positief pasiënte het ‘n aansienlike verswakte immuunstelsel, wat hul hoogs vatbaar tot opportunistiese infeksies maak, en dus, addisionele behandeling benodig. Cryptococcal meningitis en orofaringeale kandidiase word met orale flukonasool behandel. As gevolg van middeling in algemene metaboliese paaie is daar ‘n groot moontlikheid van middel-middel interaksies tussen flukonasool en die antiretrovirale (ARV) middels, efavirenz, nevirapine, en lopinavir/ritonavir. Die uitkomste hiervan mag tot die ontwikkeling van nadelige middel-middel interaksies of middelweerstandigheid en mislukte behandeling lei. Doel: Die primêre doel van hierdie tesis was om die effek van flukonasool op die farmakokinetika van efavirenz, nevirapine en lopinavir/ritonavir in HIV geïnfekteerde pasiënte met gediagnoseerde cryptococcal meningitis en orofaringeale kandidiase te evalueer. Metodes: Die studie was met 80 HIV-positief, behandeling-ervare volwassenes (≥18 jaar) onderneem. Voorafgenoemde was in drie verskillende buitepasiëntklinieke in die Wes-Kaap behandel. Pasiënte was volgens ARV regimen en die gebruik van flukonasool, of dan nie, verder verdeel. ‘n Beperkte steekproef ontwerp was gebruik, en ooreenstemmende ARV serum konsentrasies is deurgevestigde HPLC en GC metodes vasgestel. Flukonasool serum konsentrasies was deur ‘n nuutontwikkelde HPLC metode vasgestel. Pasiëntkenmerke, gepaardgaande medikasie, kliniesetoets data en ARV serum konsentrasies was by ‘n NONMEM genereerde, een-kompartement, oop farmakometriese model met eerste orde eliminasie ingesluit om enige middel interaksies tussen flukonasool en die bestudeerde ARVs op te tel. Die sekondêre uitkomste was om vas te stel watter pasiënt kenmerke ARV farmakokinetika beïnvloed. Resultate:Uit 80 buitepasïente was ‘n totaal van 276 ARV serum monsters (137 efavirenz, 67 nevirapine en 72 lopinavir) vir farmakokinetiese evaluasie gekollekteer. Efavirenz opruiming was met ras gekorreleer asook gepaardgaande gebruik van rifampisien. Geen betekenisvolle ko-variante was in die nevirapine model vasgestel nie. In die lopinavir model het die gepaardgaande gebruik van clotrimazole en die anti-tuberkulose kombinasie isoniazied, pyrazinamied en rifampisien, lopinavir opruiming verminder. Bespreking: In hierdie studie is geen betekenisvolle effekte van flukanosool op die farmakokinetika van enige van die bestudeerde ARVs waargeneem nie. Afwisselende efavirenz plasma konsentrasies in verskillende etniese populasies mag aan verskille in geenuitdrukking, veral CYP2B6, toegeskryf word. Kleurling pasïente het betekenisvolle verlaagde efavirenz serum konsentrasies getoon (56.8% verlaging in opruiming). Hierdie bevinding is nog nooit voorheen in die Suid-Afrikaanse konteks beskryf nie. Alhoewel geslag nie ‘n beduidende ko-variant in die nevirapine model was nie, het vroulike pasïente geneig om hoer nevirapine serum konsentrasies te hê. TB behandeling, in alle pasïente wat lopinavir ontvang het, het uit die volgende kombinasie bestaan: isoniazied, pyrazinamied en rifampisien, elk met hul eie effekte op CYP isoensieme. Die presiese bydra van elke middel in die uiteindelike verlaging (46.4%) in lopinavir opruiming kan dus nie vasgestel word nie. Gevolgtrekking: Gegewe die beperkings van die steekproef in die huidige studie, kon geen statistiese beduidende effek van flukonazool op die farmakokinetika van die betrokke ARVs gedemonstreer word nie. ‘n Retrospektiewe analise van die data het gewys dat verskeidene ko-faktore die farmakokinetika van die betrokke ARVs beïnvloed. Hierdie data moet in ‘n prospektiewe studie bevestig word omdat die geidentifiseerde covariates die bestuur van MIV-positiewe pasiente in die Suid-Afrikaanse konteks te verbeter.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: Cancer is an emerging health problem in South Africa, with breast cancer being one of the leading cancers affecting women globally. Therefore, there is a need to find novel targets to improve the therapeutic options for these patients. A recently proposed target is the mitogen-activated protein kinase phosphatase-1 (MKP-1). Studies have suggested that mitogen-activated protein kinase phosphatases are involved in the development of cancer and play an important role in the response of cancer cells to chemotherapy. Additionally, numerous studies have indicated that there is increased expression of MKP-1 in breast cancers where its over-expression is proposed to be a significant mediator in chemo-resistance. We propose that inhibition of MKP-1 will increase the cytotoxic effect of doxorubicin in breast cancer cells, thus making the cells more responsive to treatment leading to increased cell death through autophagy and apoptosis. Methods: In MDA-MB231 cells, MKP-1 was inhibited using sanguinarine or MKP-1 siRNA and this was compared to a known inducer of MKP-1, dexamethasone. MDA-MB231 cells were treated with doxorubicin alone or in combination with MKP-1 inhibitors or an inducer. Following treatment, cell death was determined by trypan blue and a caspase glo assay as well as with western blotting. Autophagy was determined by western blotting and flow cytometry. LC3 and p62 were used as markers of autophagy and caspase 3 and PARP as apoptosis markers. Likewise, the level of MKP-1 expression under conditions of MKP-1 induction, inhibition or silencing was evaluated by means of western blotting. C57BL6 tumour bearing mice was used to analyse apoptosis and autophagy in vivo under conditions of MKP-1 inhibition, using sanguinarine, together with doxorubicin treatment. Western blotting was used to determine levels of caspase 3, LC3, p62 and MKP-1 expression. Results and discussion: A concentration and time curve indicated that 5 μM doxorubicin reduced cell viability in the MDA-MB231 cells significantly after 24 hours of treatment. MKP-1 expression was significantly reduced with sanguinarine and MKP-1 siRNA. Furthermore, our results indicate a significant increase in apoptosis in MDA-MB231 cells when treated with doxorubicin, under conditions of MKP-1 inhibition or MKP-1 silencing. Also, an increase in autophagic activity was observed following treatment with doxorubicin in combination with sanguinarine. Whole excised tumours of C57BL6 mice also showed an increase in apoptosis and autophagy following treatment with sanguinarine in combination with doxorubicin. This indicates that the inhibition of MKP-1 with sanguinarine sensitized the MDA-MB231 cells and E0771 cell tumours to doxorubicin-induced-apoptosis through a mechanism involving autophagy. Conclusion: This is an encouraging finding that could hopefully be used in future studies to overcome doxorubicin-resistance in breast cancer cells overexpressing MKP-1. Targeting MKP-1 can have potential therapeutic benefits for breast cancer patients by making chemotherapy more effective. Sanguinarine thus has potential to be developed as a clinically relevant inhibitor of MKP-1 which could provide a novel avenue for therapeutic intervention in combination with chemotherapy in breast cancer patients. / AFRIKAANSE OPSOMMING: Inleiding: Kanker is 'n vinnig groeiende gesondheidsprobleem in Suid-Afrika, met borskanker as een van die vernaamste kankers wat vroue wêreldwyd raak. Daar is dus 'n behoefte aan nuwe terapeutiese opsies vir hierdie pasiënte en mitogeen-geaktiveerde proteïenkinase fosfatase-1 (MKP-1) is onlangs voorgestel as ‘n moontlike teiken. Verskeie studies toon dat mitogeen-geaktiveerde proteïenkinase fosfatases betrokke is by die ontwikkeling van kanker en ook belangrike rolspelers is in die reaksie van kanker op chemoterapie. Daarbenewens toon talle studies dat daar verhoogde MKP-1 uitdrukking in borskanker is, asook dat dit ‘n belangrike bemiddelaar is vir die weerstand wat borskanker teen chemoterapie bied. Ons het dus voorgestel dat die inhibisie van MKP-1 die sitotoksiese effek van doxorubicin op borskanker selle sal verhoog; sodoende sal die kanker selle beter reageer op behandeling en dit sal dus lei tot verhoogde seldood deur autofagie en apoptose. Metodes: MKP-1 is geïnhibeer met behulp van sanguinarine of MKP-1 siRNA in MDA-MB231 selle en dit is vergelyk met 'n bekende MKP-1 induseerder, dexamethasone. MDA-MB231 selle is met doxorubicin alleen behandel of in kombinasie met MKP-1 inhibeerders of ‘n induseerder. Seldood is bepaal deur middel van ‘n trypan blou en kaspase toetsingsmetode, asook met die westelike kladtegniek. Autofagie is bepaal deur westelike kladtegniek en vloeisitometrie. LC3 en p62 is gebruik as merkers van autofagie en kaspase 3 en PARP is as apoptose merkers gebruik. MKP-1 uitdrukking is geëvalueer deur middel van westelike kladtegniek. C57BL6 muise met kankeragtige gewasse is gebruik om apoptose en autofagie in vivo te ondersoek. MKP-1 is geïnhibeer met sanguinarine en die muise is behandel met ‘n kombinasie van sanguinarine en doxorubicin. Kaspase 3, LC3, p62 en MKP-1 uitdrukking is bepaal deur middel van die westelike kladtegniek. Resultate en bespreking: ‘n Konsentrasie en tyd kurwe het aangedui dat 5 μM doxorubicin die MDA-MB231 selle se lewensvatbaarheid aansienlik verminder het na 24 uur. MKP-1 uitdrukking is ook aansienlik verminder met sanguinarine en MKP-1 siRNA. Verder dui die resultate op 'n beduidende toename in apoptose in MDA-MB231 selle na behandeling met doxorubicin onder toestande van MKP-1 inhibisie. 'n Toename in autofagiese aktiwiteit is waargeneem na behandeling met doxorubicin en sanguinarine. Die kankeragtige gewasse van die C57BL6 muise toon ook 'n toename in apoptose en autofagie na behandeling met sanguinarine en doxorubicin. Hierdie resultate dui daarop dat die inhibisie van MKP-1 met sanguinarine die MDA-MB231 selle en E0771 sel gewasse gesensitiseer het tot doxorubicin-geïnduseerde apoptose deur middel van ‘n meganisme wat autofagie insluit. Gevolgtrekking: Hierdie bevinding kan hopelik in toekomstige studies gebruik word om doxorubicin weerstand te oorkom in borskanker selle waar MKP-1 verhoog is. Deur MKP-1 te teiken, kan dit lei tot potensiële terapeutiese voordele vir borskanker pasiënte en sodoende kan dit chemoterapie meer effektief maak. Sanguinarine het dus die potensiaal om ontwikkel te word as ‘n klinies relevante inhibeerder van MKP-1 wat sodoende kan dien as terapeutiese intervensie in kombinasie met chemoterapie vir borskanker pasiënte.

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: Dichloro-diphenyl-trichloroethane (DDT) was extensively used in agriculture pest control and is still used for indoor residual spraying to control malaria. The lipophylicity of DDT and its breakdown product dichloro-diphenyl-dichloroethylene (DDE) dictates that they associate with membranes, lipids and hydrophobic proteins in the biological environment. Their poor degradable nature causes DDT and DDE to persist for decades in the environment and in individuals who are or were in contact with the pesticide. In many countries the synchronised resistance of the mosquito vector to insecticides and the malaria parasite towards antimalarial drugs led to a drastic rise in malaria cases and to malaria epidemics. This study assesses the influence of low level exposure of DDT and DDE on chloroquine (CQ) resistance of the dire human malaria parasite, Plasmodium falciparum. The in vitro activity of p,p’-DDT and p,p’-DDE towards blood stages of chloroquine sensitive (CQS) P. falciparum D10 and chloroquine resistant (CQR) P. falciparum Dd2 was determined using two complementary in vitro assays (Malstat and SYBR Green 1). The 50% inhibition concentrations (IC50s) of p,p’-DDT and p,p’-DDE were found to be ±14 to 38 μM (5-12 μg/mL) and highly similar towards CQS and CQR P. falciparum strains. This result indicated that the proteins involved in CQ resistance have no effect on the activity of the insecticide DDT and it breakdown product DDE. In order to assess the influence of DDT and DDE on CQ activity, in vitro fixed ratio drug combination assays were performed, as well as isobologram analysis. We found that CQ works in synergy with p,p’-DDT and p,p’-DDE against CQS P. falciparum D10. However, both p,p’-DDT and p,p’-DDE were antagonistic toward CQ activity in CQR P. falciparum Dd2. This indicated that p,p’-DDT and p,p’-DDE do have an effect on CQ resistance or on the action of CQ via a target other than hemozoin polymerization. The observation of reciprocal synergism of p,p’-DDT and p,p’-DDE with CQ against CQS D10 and antagonism against CQR Dd2 strain is highly significant and strongly indicates selection of CQ resistant strains in the presence of p,p’-DDT and p,p’-DDE. People who have low levels of circulating DDE and/or DDT could be at a high risk of contracting CQR malaria. However, medium term (nine days) DDE exposure of CQS P. falciparum D10 did not induce resistance, as no significant change in activity of CQ, p,p’-DDT and p,p’-DDE towards blood stages the CQS strain was observed. This exposure was, however, shorter than expected for a malaria infection and would be addressed in future studies. From our results on the interaction of CQ with p,p’-DDT and p,p’-DDE, it was important to assess the residual DDT and DDE variable and how much of residual p,p’-DDT and/or p,p’- DDE would enter into or remain in the different compartments (the RPMI media, erythrocytes and infected erythrocytes) over time. In combination with liquid-liquid extraction, we developed a sensitive GC-MS analyses method and a novel HPLC-UV analysis method for measuring DDT and DDE levels in malaria culturing blood and media. Whilst the HPLC-UV method was relatively cheaper, faster, and effective in determining high DDT and DDE concentrations, the optimised GC-MS method proved to be effective in detecting levels as low as 78 pg/mL (ppt) DDE and 7.8 ng/mL (ppb) DDT in biological media. Using both the HPLC and GC-MS methods we observed that malaria parasites influence distribution of the compounds between the erythrocytic and media fractions. P. falciparum D10 infection at ±10% parasitemia lead to must faster equilibration (less than 8 hours) between compartments. Equimolar distribution of p,p’-DDE was observed, but the parasites lead to trapping of the largest fraction of p,p’-DDT in the erythrocyte compartment. These results indicate that a substantial amount would reach the intra-erythrocytic parasite and could influence the parasite directly, possibly leading to either synergistic or antagonistic drug interactions. This study is the first to illustrate the “good and bad” of the insecticide DDT in terms of CQ resistance and sensitivity toward the human malaria parasite P. falciparum. These results will hopefully have an important influence on how future policies on malaria control and treatment particularly in endemic areas will be addressed and could also have an impact on the anti-malarial drug discovery approach. / AFRIKAANSE OPSOMMING: Dichlorodifenieltrichloroetaan (DDT) is op groot skaal in landbouplaagbeheer gebruik en word nog steeds gebruik vir binnenshuise oppervlakbespuiting om malaria te beheer. Die lipofilisiteit van DDT en sy afbraakproduk dichlorodifenieldichloroetileen (DDE) dikteer dat hulle met membrane, lipiede en hidrofobiese proteïene in die biologiese omgewing assosieer. Stadige afbraak veroorsaak dat DDT en DDE vir dekades in die omgewing agterbly, asook in individue wat in kontak is, of was met die insekdoder. In baie lande het gesinkroniseerde weerstand van die muskietvektor teenoor insekdoders en die malariaparasiet teenoor antimalariamiddels gelei tot 'n drastiese styging in malariagevalle en tot malariaepidemies. In hierdie studie word die invloed van lae vlak blootstelling van DDT en DDE op chlorokien (CQ) weerstand van die mens malariaparasiet, Plasmodium falciparum, geëvalueer. Die in vitro aktiwiteit van p,p'-DDT en p,p'-DDE teenoor die bloedstadia van chlorokiensensitiewe (CQS) P. falciparum D10 en chlorokien-weerstandbiedende (CQW) P. falciparum Dd2 is bepaal deur gebruik te maak van twee komplementêre in vitro toetse (Malstat en SYBR Groen toetse). Die 50% inhibisie konsentrasies (IC50s) van p,p'-DDT en p,p'-DDE is bepaal as ±14 to 38 μM (5-12 μg/mL) en was hoogs vergelykbaar tussen CQS en CQW P. falciparum stamme. Hierdie resultaat het aangedui dat die proteïene betrokke by CQ weerstand geen effek op die aktiwiteit van die insekdoder DDT en die afbraakproduk DDE het nie. Om die invloed van DDT en DDE op CQ aktiwiteit te evalueer, is die aktiwiteit van kombinasies van die verbindings in vaste verhoudings getoets, tesame met isobologram ontleding. Ons het gevind dat CQ sinergisties saam met p, p'-DDT en p, p'-DDE teen CQS P. falciparum D10 werk. Daarteenoor het beide p, p'-DDT en p, p'-DDE antagonistiese werking getoon teenoor CQ aktiwiteit met CQW P. falciparum Dd2 as teiken. Dit het aangedui dat p,p'-DDT en p, p'-DDE wel 'n invloed op CQ weerstand het of ‘n aktiwiteit van CQ, anders as hemozoin polimerisasie, beïnvloed. Die waarneming van resiproke sinergisme en antagonisme van p, p'-DDT en p, p'-DDE in kombinasie met CQ teenoor die CQS D10 en CQW DD2 stamme respektiewelik, is hoogs betekenisvol en dui op seleksie van CQweerstandige stamme in die teenwoordigheid van p, p'- DDT en p, p'-DDE. Mense wat lae vlakke van sirkulerende DDE/DDT het, het dus 'n hoër risiko om CQW malaria te kry. Verder is gevind dat medium termyn (nege dae) DDE blootstelling van CQS P. falciparum D10 nie weerstand nie veroorsaak nie, want geen beduidende verandering in die aktiwiteit van CQ, p,p'-DDT en p,p'-DDE teenoor die bloed stadiums van die CQS stam is waargeneem nie. Hierdie blootstelling is egter korter as in 'n malaria-infeksie en sal verder bestudeer word in toekomstige studies. Vanuit die interaksie resultate van CQ met p, p'-DDT en p, p'-DDE was dit belangrik om die residuele DDT en DDE veranderlike te evalueer, asook die distribusie van p,p'-DDT en p,p'- DDE tussen die verskillende kompartemente (die kultuurmedium, eritrosiete en geïnfekteerde rooibloedselle) oor verloop van tyd. In kombinasie met vloeistof-vloeistof ekstraksie, het ons 'n sensitiewe GC-MS en nuwe HPLC-UV analisemetode ontwikkel vir die meet van DDT en DDE-vlakke in bloed (normale en geïnfekteerde eritrosiete) en die kultuurmedium. Terwyl die HPLC-UV metode relatief goedkoper, vinniger en effektief in die bepaling van hoë DDT en DDE-konsentrasies is, was die geoptimaliseerde GC-MS metode doeltreffend in die opsporing van vlakke so laag as 78 pg/mL (dpt) DDE en 7.8 ng/mL (dpb) DDT in biologiese media. Met behulp van beide die HPLC-UV en GC-MS metodes is waargeneem dat die malariaparasiet die ekwilibrasie van die verbindings tussen die eritrosiet- en media kompartemente beïnvloed. P. falciparum D10 infeksie met ± 10% parasitemia lei tot vinniger ekwilibrasie (minder as 8 uur) tussen die kompartemente. Ekwimolêre verspreiding van p,p'- DDE is waargeneem, maar die parasiete het die grooste fraksie van p,p'-DDT in die eritrosiet kompartement vasgevang. Hierdie resultate wys dat 'n aansienlike fraksie die intraeritrositiese parasiet kan bereik en sodoende die parasiet direk kan beïnvloed en moontlik kan lei tot sinergistiese of antagonistiese middel interaksies. Hierdie studie is die eerste om die "goed en sleg" van die insekdoder DDT in terme van CQ weerstand en sensitiwiteit teenoor die menslike malariaparasiet P. falciparum te illustreer. Hierdie resultate sal hopelik 'n belangrike invloed hê op die toekomstige beleid oor die beheer van malaria en behandeling, veral in endemiese gebiede, en mag ook 'n impak hê op die antimalariamiddel navorsing.

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.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: In this dissertation, I grapple with the problem of global drug resistance and moral responsibility which, as far as I am aware, has so far not been presented as a topic of ethical inquiry. It represents a conundrum involving three major factors: microbial adaptation and change, human social factors and environmental changes. Drug resistance is a phenomenon in which certain microorganisms, when exposed to antimicrobial agents, may acquire the beneficial trait of drug resistance which ensures a better potential for their survival. The acquired trait of drug resistance I argue renders such microorganisms 'supra-natural '. Supra-natural is a term I coin for entities that have been imposed upon nature by human design; they do not follow the natural evolutionary processes of adaptation and change. Drug resistance is classified as an emerging infectious disease. Human social factors and environmental change (particularly population growth, density and consumerist practices) enhance the rise of emerging infectious diseases. Through such increasing destructive practices, stress is placed on the environment. Environmental stress facilitates the rise of new and old infectious diseases and the spread of drug resistant supra-natural microorganisms. Thus, our ability to treat successfully illnesses and injuries in humans, animals and plants is increasingly impaired. Morally, we are responsible for the problem of global drug resistance. Drug resistant microorganisms exist in nature and concerning this, we can do nothing. At best, we can only try to control the problem using prudential measures. The problem of global drug resistance represents both a biomedical ethical and an environmental ethical issue. Is there a way out of the human-nature debate? Through Bryan Norton's enlightened anthropocentrism, I identify the ways in which his thesis may be applied to the problem of human and environmental concerns and show its applicability in broadening the parameters of biomedical ethics education to include environmental concerns. Key words: biomedical ethics, environmental ethics, drug resistance, Supra-natural' microorganisms, ethics education, enlightened-anthropocentrism. / AFRIKAANSE OPSOMMING: In hierdie proefskrif bespreek ek die probleem van die verskynsel dat mikroorganismes op 'n globale skaal weerstand begin bied teen mediese middels (globale middel-weerstandigheid) en die morele verantwoordelikheid wat dit oproep - 'n probleem wat, na my beste wete, nog nooit aangebied is as 'n tema van etiesfilosofiese ondersoek nie. Dit verteenwoordig 'n kompleks van drie belangrike oorwegings: mikrobiese aanpassings en veranderinge, menslike sosiale faktore, en omgewingsveranderinge. Middel-weerstandigheid is 'n verskynsel waarin sekere mikro-organismes, wanneer hulle blootgestel word aan antimikrobiese agente, die (vir hulself) voordelige kenmerk kan bekom van weerstandigheid teen die middel; iets wat 'n beter potensiaal vir hul eie oorlewing verseker. Hierdie bekomde kenmerk (middel-weerstandigheid) maak, volgens my argument, sulke mikro-organismes 'supra-natuurlik'. Supra-natuurlik is 'n term wat ek munt vir entiteite wat aan die natuur blootgestel is as gevolg van menslike ontwerp; hulle volg nie die natuurlike evolusionêre prosesse van adaptasie en verandering nie. Middel-weerstandigheid word geklassifiseer as 'n opkomende aansteeklike siekte. Menslike sosiale faktore en omgewingsveranderinge (veral bevolkingsgroei, -digtheid and verbruikerspraktyke ) vergroot die opkoms van aansteeklike siektes. Deur sodanige toenemende destruktiewe praktyke word stres geplaas op die omgewing. Omgewingstres fasiliteer die opkoms van nuwe en ou aansteeklike siektes asook die verspreiding van weerstandige supra-natuurlike mikro-organismes. Ons vermoë om siektes en beserings van mense suksesvol te behandel, word gevolglik toenemend ondermyn. Moreel gesproke is ons verantwoordelik vir die probleem van globale middelweerstandigheid. Middel-weerstandige mikro-organismes bestaan in die natuur, en aan daardie feit as sodanig kan ons niks doen nie. Ons kan, ten beste, probeer om die probleem te beheer deur middel van verstandige maatreëls. Die probleem van globale middel-weerstandigheid verteenwoordig sowel 'n biomedies-etiese as 'n omgewingsetiese kwessie. Is daar 'n uitweg uit die mens-natuur debat? Ek identifiseer, met 'n beroep op Bryan Norton se swak antroposentrisme, maniere waarop sy tese toegepas sou kon word op die probleem van menslike en omgewingsoorgwegings Ek wys ook op die toepaslikheid daarvan vir die verbreding van die parameters van biomediese etiek-opvoeding ten einde omgewingsoorwegings deel van lg. te maak. Kembegrippe: biomediese etiek, omgewingsetiek, middel-weerstandigheid, 'Supra- . natuurlike' mikro-organismes, etiek-opvoeding, swak antroposentrisme.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy