published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The global emergence of drug resistance is posing increasing difficulties in the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs) are regarded as having a pivotal role among the antimicrobial agents in multidrug regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of TB therapy. The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon the bactericidal effect and prevention of the emergence of resistance. In the first part of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90, AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as a promising agent for the treatment of MDR-TB. Molecular tests on resistance genes are reliable and rapid technology for diagnosis of drug-resistant TB which facilitates timely patient management and public health control of TB. In the second part of the study, the feasibility of a PCRsequencing assay for the examination of mutations in the quinolone-resistance-determining- region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr) Mycobacterium tuberculosis in direct clinical specimens was evaluated. As determined by gyrA QRDR DNA sequencing analysis, complete concordance of phenotypic and genotypic outcomes was demonstrated. The results indicate that the molecular assay is an accurate and effective method for the diagnosis of FQr TB and allows identification of mixed resistant variants in the same patient. GyrA mutations that associated with FQr in clinical isolates of M. tuberculosis were clustered in hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory specimens by using the PCR-DNA sequencing assay. In the third part of this study, the functional effect of the Ala74Ser mutant was verified through study of the DNA supercoiling inhibitory activities of OFX and MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations (IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than the wild-type H37Rv reference strain, and results correlated well with their phenotypic drug susceptibilities. Besides, a combination of gyrA mutations (Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3 helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer. Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative drug binding pocket, and leads to the reduction of the binding affinity of FQ due to the distance effect. This is the first report verifying that gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Iron is an essential element that plays a role in the process of respiration, oxygen transport and as a principle cofactor to several enzymes. Iron homeostasis is a finely regulated process since excess levels become toxic to healthy cells via the production of reactive oxygen species. A plethora of genes that control several key points throughout this regulatory process have been identified. Research focusing on changes in expression levels and downstream functional effects of these genes has become increasingly important over the past decade. One area of particular interest has emerged since a link between iron status and host response to Mycobacterium tuberculosis infection was discovered. Although the prevalence of Tuberculosis has decreased across the globe with the exception of Africa and parts of Europe, the mortality rate remains high. Therefore, research that focuses on understanding an individual’s predetermined susceptibility to TB infection at the genetic level could provide health care practitioners with the tools required to identify and educate at-risk individuals prior to TB infection. RT-qPCR was utilised to determine expression profiles for eight iron genes (CP, CYBRD1, FTH, FTL, LTF, HFE, HMOX1, and SCL40A1) normalised to three reference genes (ACTB, GUSB, and RPL37A1). Up-regulation is demonstrated in the TB group for transcript levels recorded for CYBRD1, HFE, HMOX1, and SLC40A1. Several measured serum parameters including conjugated, unconjugated, total bilirubin, and total protein were increased in the TB group while albumin was significantly lower in this group. Correlation analysis demonstrated that a positive correlation exists between transferrin saturation and iron and a negative correlation exists between transferrin and ferritin levels. Individuals categorised with low serum iron levels demonstrated lower CP/GUSB levels and higher HMOX1/GUSB levels. Individuals categorised with low transferrin saturation levels demonstrated higher FTL/GUSB and SLC40A1/GUSB levels and lower CP/GUSB. Results from this study provide further evidence for the relationship between iron status and TB infection rates, although protein studies are required to confirm these results. The data obtained illustrate the important role that these profiles and iron parameters may play in the clinical field when identifying at-risk individuals. Further investigation that focuses on which gene profile and parameter combinations show the most distinctive utility in the clinical setting is warranted. / AFRIKAANSE OPSOMMING: Yster is ‘n noodsaaklike element wat ‘n rol speel in die proses van respirasie en die vervoer van suurstof en ook ‘n belangrike ko-faktor vir verskeie ensieme is. Yster homeostase is op ‘n fyn manier gereguleer omdat oormatige vlakke toksies kan wees vir gesonde selle wanneer reaktiewe suurstofspesies geproduseer word. ‘n Magdom gene wat verskeie sleutelpunte in hierdie proses kontroleer is voorheen identifiseer. Navorsing wat fokus op die veranderinge in geenuitdrukkingsvlakke en die funksionele gevolge daarvan het oor die afgelope dekade toenemend belangrik geword. Een gebied van spesifieke belang het na vore gekom nadat ‘n verband tussen ystervlakke en die manier waarop die immuunstelsel reageer op Mycobacterium tuberculosis infeksie, ontdek is. Alhoewel die voorkoms van Tuberkulose wêreldwyd, behalwe in Afrika en sekere dele van Europa, afgeneem het, bly die sterftesyfer hoog. Daarom kan navorsing wat daarop fokus om ‘n individu se voorafbepaalde vatbaarheid vir TB-infeksie op die genetiese vlak te verstaan dalk aan gesondheidswerkers die regte instrumente verskaf om hoë-risiko individue te identifiseer en op te voed voordat hulle TB ontwikkel. RT-qPKR is gebruik om die geenuitdrukkingsvlakke van agt ystergene, wat met drie verwysings-gene (ACTB, GUSB, en RPL37A1) genormaliseer is, te bepaal. ‘n Toename in die uitdrukkingsvlakke van CYBRD1, HFE, HMOX1, en SLC40A1 is in die TB-groep waargeneem. Die bloedvlakke van verskeie parameters insluitend gekonjugeerde, ongekonjugeerde, totale bilirubin, en totale proteïen was hoër in die TB-groep, terwyl albuminvlakke laer was in hierdie groep. Korrelasie-analise het ‘n positiewe korrelasie tussen transferrin-versadiging en yster getoon, terwyl daar ‘n negatiewe korrelasie tussen transferrin- en ferritinvlakke gevind is. Individue met lae ystervlakke het laer CP/GUSB-vlakke en hoër HMOX1/GUSB-vlakke getoon. Individue met lae transferrin-versadiging het hoër FTL/GUSB- en SLC40A1/GUSB-vlakke en laer CP/GUSB-vlakke getoon. Resultate uit hierdie studie verskaf verdere getuienis dat daar ‘n verwantskap tussen ystervlakke en TB-infeksiekoerse bestaan, alhoewel proteïenstudies nodig is om hierdie resultate te bevestig. Die data dui op die belangrike rol wat hierdie profiele en ystervlakke in die kliniese veld mag speel in die identifisering van hoë-risiko individue. Verdere ondersoek, gefokus op watter geenprofiel en parameterkombinasies die grootste nut in die kliniese omgewing bied, is geregverdig.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / According to the Namibia National Tuberculosis Control Programme (NTCP) report of 2008, Namibia has one of the highest TB infection rates in the world with a case notification rate of 748/100,000. Rapid, specific and sensitive diagnosis of Mycobacterium tuberculosis (MTB) is needed for correct TB patient management. One of the aims of this study was thus to compare direct microscopy with two rapid molecular diagnostic tools (viz. GeneXpert MTB/RIF and Hain Genotype® MTBDR plus assay) for the identification of MTB from samples collected from the Khomas Region, Windhoek, Namibia. Only patients with positive TB sputum collected at the clinics and health facilities in the Khomas Region, Windhoek were eligible for the study. Three hundred and eighty-four samples were confirmed acid-fast positive by utilising the auramine staining method. The rifampicin (RIF) resistance profile detected by both molecular techniques was then compared for characterisation of the samples as drug resistant. Lastly, participants completed a survey, which included questions related to demographic and epidemiological data. Demographic data included patient age, gender, region of residence and history of treatment. The data was collected using a structured questionnaire and was captured in an Excel spreadsheet. It was then imported into Statistical Package for Social Sciences (SPSS) Version 25 for data analysis. A memorandum of understanding was also signed with the Namibia Institute of Pathology (NIP) to obtain permission to use their samples and the equipment at their site.

Thesis (MScMedSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Application of molecular fingerprinting highlights transmission as the driving force behind the drug resistant epidemic in South Africa. Different strains dominate within different geographical regions, which is a reflection of micro-epidemics of drug resistance in the different regions. Cluster analysis shows that strains within the same strain family are different. The Beijing drug resistant strain family is the most dominant strain family (31%) in the Western Cape and of particular concern is the highly transmissible Beijing cluster 220 strain in the Western Cape communities. This cluster is widespread in the region and was previously identified in a MDR outbreak in a high school in Cape Town. Results suggest that the spread of Beijing drug resistant cluster 220 in the community was due to a combination of acquisition of drug resistant markers and transmission. This study also indicate that atypical Beijing can acquire drug resistance and become fit amongst HIV infected individuals. This is contrary to believe that atypical Beijing strains are not frequently associated with drug resistance and are attenuated. This implies that HIV levels the playing field for all drug resistant strains. Mechanisms leading to the evolution of MDR-TB and XDR-TB in a mine setting with a wellfunctioning TB control program which exceeds the target for cure rates set by the WHO were investigated. Despite the excellent control program, an alarming increase in the number of drug resistant cases was observed in 2003 and subsequent years. Phylogenetic analysis shows sequential acquisition of resistance to first and second-line anti-TB drugs leading to the development of MDR and XDR-TB. Contact tracing indicate extensive transmission of drug resistant TB in the shafts, hospital and place of residence. This study shows that despite exceeding the WHO cure rate target, it was not possible to control the spread and amplification of drug resistance. In summary, as a top priority, future TB control plans need to address diagnostic delay more vigorously. / AFRIKAANSE OPSOMMING: Molukulêre tegnieke toon transmissie as die hoofrede vir die toename in die anti-tuberkulose middelweerstandigheid epidemie in Suid-Afrika. Die verskillende Mikobakterium tuberkulose rasse wat domineer in verskillende areas is ‘n refleksie van middelweerstandige mikro-epidemies in verskillende gebiede. Analise van identiese rasgroepe demonstreer dat ras families bestaan uit verskillende rasse. Die Beijing middelweerstandige rasfamilie is die mees dominante familie in die Wes-Kaap (31% van monsters van middelweerstandige families) en van spesifieke belang is die hoogs oordraagbare Beijing 220 groep. Hierdie groep is die mees wydverspreide groep in die studie area en was voorheen geïdentifiseer tydens ‘n meervoudige middelweerstandige uitbreking in ‘n hoërskool in Kaapstad. Die resultate dui aan dat die Beijing middelweerstandige groep 220 in die gemeenskap versprei as gevolg van ‘n kombinasie van middelweerstand verwerwing en transmissie. Hierdie studie dui verder aan dat die atipiese Beijing ook middelweerstandigheid kan verwerf en hoogs geskik is vir infeksie veral in MIV geïnfekteerde individue. Hierdie data is in teenstelling met die algemene denke dat atipiese Beijing nie gereeld geassosieer word met middelweerstandigheid nie en dat dit dikwels geattenueer is. Dit beteken dat MIV die hoof faktor is wat alle middelweerstandige rasse kans gee om te versprei. Hierdie studie het die meganisme wat lei tot die evolusie van middelweerstandigheid en “XDRTB” in die myne ondersoek. Die myn besit ‘n goeie funksioneerde tuberkulose kontrole program wat alreeds die Wêreld Gesondheids Organisasie se mikpunt vir tuberkulose genesing oortref. Ten spyte van ‘n uitstekende tuberkulose kontrole program, is daar ‘n bekommerenswaardige toename in die aantal middelweerstandige tuberkulose gevalle waargeneem in 2003 en in die daaropvolgende jare. Filogenetiese analise wys dat opeenvolgende verwerwing van middelweerstandigheid teen eerste en tweede vlak anti-tuberkulose middels gelei het tot die ontwikkeling van meervoudige middelweerstandigheid en “XDR-TB”. Die opsporing van kontakpersone om transmissie te bewys dui aan dat transmissie van middelweerstandige tuberkulose in die werk plek, hospitaal en woon plek plaasvind. Hierdie studie wys dat ongeag die feit dat die Wêreld Gesondheids Organisasie se genesings verwagtinge oortref is, dit steeds onmoontlik was om die verspreiding en amplifisering van middelweerstandigheid te beheer. ‘n Top prioriteit vir tuberkulose kontrole planne in die toekoms behoort die vertraging van diagnose sterk aan te spreek.