Submitted in fulfillment of the requirements for the degree of Master’s of Technology: Radiography, Durban University of Technology, Durban, South Africa, 2015. / Introduction Tuberculosis remains a leading cause of death, second to the Human Immunodeficiency Virus. The risk of latent tuberculosis infection and active tuberculosis disease is a known occupational hazard. In South Africa, a high tuberculosis burden country, the potential of Mycobacterium tuberculosis transmission to health care workers is high. This includes diagnostic radiographers and other radiology staff working in radiology departments. Purpose of the Study This study aimed to investigate the association of demographic and occupational factors with latent tuberculosis infection in radiology staff in public sector hospitals of the eThekwini Health District. Methodology This cross-sectional study was conducted from 26 February 2013 to 07 June 2013. Quantitative methods were used to test for associations of demographic and occupational factors with latent tuberculosis infection in participants. A sample size of 181 participants for an estimated population of 340 radiology staff was recommended at the proposal stage. The study consisted of two phases; the questionnaire survey (phase one) and the administration of a two-step tuberculin skin test (phase two). Data was obtained with regard to demographics, occupational history, social behaviours, medical history; and family and home histories. Demographic and occupational associations with latent tuberculosis infection were made in relation to the size of the first tuberculin skin test induration. Frequency distributions were developed to describe data categories. Pearson’s and Spearman rho’ correlation coefficients were used to test for correlations between the independent variables. The chi-square test was used to determine associations between the categorical independent variables and the dependent variable. Bivariate analyses were performed using these tests. The multivariate analysis was performed using logistic and linear regression on the dependent variable. Results A total of 182 questionnaires were returned from approximately 280 radiology staff. At the outset, all doctors working in the radiology department had to be excluded due to numerous failed attempts to enlist their participation. Fifty-three (29.12 percent) participants were excluded from phase one of the study and a further thirteen participants were excluded from phase two. The total sample was 116 participants. Of the 116 participants, 86.2 percent tested positive for latent tuberculosis infection at the first step of the two-step testing method used. One (0.86 percent) participant went on to convert at the second step, testing positive at this level. Demographic associations with latent tuberculosis infection included age (older) as an associated factor. A significant demographic association with latent tuberculosis infection was the use of alcohol (p-value 0.033 on the multivariate analysis). Occupational associations with latent tuberculosis infection included longer durations of employment. The annual income (higher income earners) displayed significant associations with latent tuberculosis infection (p-value 0.048 on the multivariate analysis). It is necessary in this study to note that participants include support personnel (lower income earners) making up 37.8 percent of the study, diagnostic radiographers making up 48.3 percent; and radiography managers/assistant managers (highest income earners) making up 13.8 percent of the study. Conclusion and recommendations The risk of transmission of Mycobacterium Tuberculosis to health care workers is a known occupational hazard. This study has described the prevalence of latent tuberculosis infection in radiology staff, at district and regional hospitals within the eThekwini Health District. With 23.62 percent of all participants already having active TB disease and 86.2 percent of the tested group displaying positive results for latent tuberculosis infection, using the tuberculin skin tests, the need for tuberculosis screening is essential. The findings of this study will be used as a health improvement mechanism for stakeholders, having identified potential gaps in medical screening in healthcare in Kwa-Zulu Natal. This study makes recommendations for the early detection of active tuberculosis infection and the monitoring of health care workers that are latently infected, thus assisting in reducing the rate of conversion of latent tuberculosis infection to active tuberculosis disease in radiology staff. This reduces long-term exorbitant costs related to health care associated infections, such as tuberculosis. It also reduces rates of transmission and cross infection to both co-workers and already immunocompromised patients, helping to curb the overall epidemic in South Africa.

Mycobacterium tuberculosis remains one of the most common worldwide causes of illness and death due to an infectious disease. The emergence of multiple and extreme-drug resistant strains has increased the need to find an effective vaccine for tuberculosis. The goal of our research group is to engineer a temperature-sensitive (TS) M. tuberculosis strain that can be used as a tool in vaccine development. One approach to create TS M. tuberculosis involves the integration of the essential gene ligA encoding a TS NAD+ dependent DNA ligase, which was taken from the psychrophilic organism Pseudoalteromonas haloplanktis. The integration and functioning of ligA was demonstrated in the fast-growing organism Mycobacterium smegmatis. This strain had a TS phenotype with growth limited to below 37°C. The strain was found to have a stable TS phenotype and did not mutate to a temperature-resistant form at a detectable level. Following experiments with the fast growing M. smegmatis, the integration of the ligA gene was attempted in slow-growing M. tuberculosis. Merodiploids of M. tuberculosis containing both the psychrophilic and the WT ligA gene in its chromosome were obtained. The second approach used for the development of TS M. tuberculosis was the directed evolution of native M. tuberculosis essential genes. An advantage of this approach is that the gene encoding the essential protein will resemble the native M. tuberculosis gene and thus will closely match the native transcriptional and translational rates. A system to screen and select for TS essential genes engineered by directed evolution was designed, where the essential gene on the chromosome of E. coli was knocked out and this gene was supplied on a conditionally replicating plasmid. As a first step in developing this directed evolution approach, a family of conditionally replicating plasmids were created and tested in an essential gene knock-out strain of E. coli. / Graduate

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2006. / The ESAT-6 gene cluster regions are duplicated 5 times in the genome of Mycobacterium tuberculosis. ESAT-6 gene cluster region 1 is the most frequently studied region as it contains RD1 (region of difference 1). RD1 is a 9.5 Kb deletion region confirmed to be involved in mycobacterial virulence and pathogenesis, and is present in virulent M. bovis strains, yet absent in all attenuated M. bovis BCG vaccine strains. The antigens CFP-10 and ESAT-6, which both evoke strong T-cell responses in experimental animals and humans, are situated in the RD1 region, and are thought to be key antigens in mycobacterial virulence. The absence of this region from the genomes of all BCG vaccine strains, led to the conclusion that the mechanism of attenuation of M. bovis BCG was due to the loss of RD1. Studies have shown that this attenuation is attributed to the loss of cytolytic activity mediated by secreted ESAT-6 (and some of the genes responsible for its secretion), which in turn results in reduced tissue invasiveness. The potent T-cell antigens ESAT-6 and CFP-10 are secreted without ordinary sec-dependent secretion signals. A study of the potential functions of the proteins encoded by the ESAT-6 gene clusters shows that most of these proteins have a potential to function in a protein-dependent ATP-binding cassette active transport system. It has been shown that ESAT-6 gene cluster region 1 is responsible for the secretion of the ESAT-6 and CFP-10 genes contained in this region, explaining the absence of any ordinary sec-dependent secretion signals in the amino acid sequences of members of this family. In order to elucidate the regulation of expression of the ESAT-6 gene cluster region 1, shown to encode for a secretion system for ESAT-6 and CFP-10 and to be involved in virulence, an operon analysis and promoter identification experiments were carried out in this study. The analysis of the ESAT-6 gene cluster region 1 showed the existence of more than one operon in this region and three constitutively-expressed promoters driving the expression of the genes in the operons. These results provide insight into the functional relationship (regulatory and secretory mechanisms) between the genes contained within ESAT-6 gene cluster region 1.None of the other four ESAT-6 gene cluster regions have been proven to also encode secretion systems. Preliminary studies indicated that the ESAT-6 gene cluster region 3 is expressed in its entirety as one single operon and a strong promoter involved in the expression of this region was identified. Mtb9.9A (the ESAT-6 antigen of the ESAT-6 gene cluster region 5) have also been shown to evoke strong T cell responses and to be secreted without any ordinary secretion signal. During the present study, we thus aimed to investigate the secretion of Mtb9.9A in order to determine whether it is also secreted by a dedicated secretion system encoded by ESAT-6 gene cluster region 5. The fact that region 5 was shown to be the last of the four duplications is important, as a positive result with this region would indicate whether the other four gene clusters share a similar secretion function. ESAT-6 gene cluster regions 2, 4 and 5 were isolated in the present study to form part of subsequent ESAT-6 gene cluster region secretion studies. Mtb9.9A was cloned, expressed and purified for antibody-generation, Resulting antibodies were used in an antigen secretion analysis. The secretion analysis entailed the integration of the isolated ESAT-6 gene cluster region 5 into the genome of M. smegmatis and investigation of the influence of the genes (contained in region 5) on the secretion of a heterologously expressed Mtb9.9A-HA-tagged fusion protein. We therefore attempted to show whether the proteins encoded by the ESAT-6 gene cluster region 5 also function together as a mycobacterial membrane-bound complex involved in protein-dependent transport and if so, whether this transport system is responsible for the active secretion of the native ESAT-6 antigen (designated Mtb9.9A) of region 5. This study opens the way for the understanding of the regulation, transport- and secretion mechanisms of important T-cell antigens of the mycobacteria, thereby giving insight into and building onto our understanding of the pathogenicity of Mycobacterium tuberculosis. A better understanding of these mechanisms could lead to the development of efficient strategies to either terminate or enhance secretion of antigens, which in turn will have an impact on drug and vaccine design and development.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one third of the world’s population and kills 1.7 million people per year. The increasing prevalence of multi- and extensively drug resistant M. tuberculosis strains means that there is an urgent need to develop new anti-TB drugs. The genome of M. tuberculosis has five copies of the ESAT-6 gene clusters (ESX-1, -2, -3, -4 and -5), which are essential for the survival (ESX-3) and pathogenicity (ESX-1 and ESX-5) of the bacterium. The ESX clusters encode for proteins which form a novel secretion system which has been shown to secreted small T-cell antigens of the esx gene family, as well as other proteins such as the PE and PPE’s. The mycosins are a family of genes situated in the ESX clusters which encode for putative subtilisin-like serine proteases. These proteins are the most conserved proteins within the five clusters. Apart from their conserved protein sequence, mycosin-3 is also an essential protein specific to the mycobacteria, which makes it an attractive potential drug target. Identifying the substrate(s) of mycosin-3 could help to understand the function of this enzyme and discover novel inhibitors from which new drugs could be designed. We hypothesize that the secreted products of the ESX system could be potential substrates for the mycosins. Specifically, we hypothesize that PE5, PPE4, esxG and esxH (all found in ESX-3) might be the substrates for mycosin-3. Mycosin-3, PE5, PPE4, esxG and esxH were thus cloned, expressed and purified respectively. The four substrates were used for protease assays using mycosin-3 as the protease. The protease-substrate mixture were subsequently separated on 2-D SDS-PAGE gels to check whether there were any cleavage of the four substrates. Although all the target fusion proteins were cloned and expressed successfully, the protease assay results showed no cleavage for any of the four substrates. Possible explanations for the failure of cleavage were: (1) impure enzyme and substrate(s); (2) inappropriate buffer conditions; (3) the hypothesized substrates might not be the substrates of mycosin-3; and (4) incorrect folding or modification of the target fusion proteins might have taken place. Future research will aim to address these possible limitations in order to fully elucidate the function and substrate specificity of mycosin-3 and to use this information for the design of novel drugs against M. tuberculosis. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat tuberkulose (TB) veroorsaak, infekteer `n derde van die wêreld se bevolking en veroorsaak die dood van 1.7 miljoen mense per jaar. Die verhoogde voorkoms van multi- en ekstensiewe middelweerstandige stamme van M. tuberculosis beteken dat daar `n ernstige nodigheid is om nuwe anti-TB middels te ontwikkel. Die genoom van M. tuberculosis het vyf kopieë van die ESAT-6 geengroepe (ESX-1, -2, -3, -4 en -5), wat essensieel is vir die oorlewing (ESX-3) en patogenisiteit (ESX-1 and ESX-5) van die bakterium. Die ESX groepe enkodeer vir proteïene wat `n nuwe uitskeidingssisteem vorm wat bewys is om klein T-sel antigene van die esx geenfamilie, sowel as ander proteïene soos die PE en PPE proteïene uit te skei. Die mycosins is `n familie gene wat in die ESX geengroepe voorkom en wat waarskynlik enkodeer vir subtilisin-agtige serine proteases. Hierdie proteïene is die mees gekonserveerde proteïene in die vyf geengroepe. Mycosin-3 is `n essensiële protein wat spesifiek in die mikobakteriëe voorkom, sodat dit `n aantreklike teiken vir die ontwikkeling van middels is. Die identifisering van die substrate van mycosin-3 kan moontlik help om die funksie van die ensiem te verstaan en om nuwe inhibeerders vir die ensiem te ontdek, wat kan lei tot die onwikkeling van nuwe middels. Ons hipotese is dat die uitgeskeide proteïene van die ESX sisteem moontlik die substrate van die mycosin proteïene kan wees. Meer spesifiek, ons hipnotiseer dat die proteïene PE5, PPE4, esxG en esxH (wat almal in ESX-3 voorkom) die substrate vir mycosin-3 kan wees. Mycosin-3, PE5, PPE4, esxG en esxH is afsonderlik gekloneer, uitgedruk en gesuiwer. Die vier substrate is gebruik vir protease proewe met mycosin-3 as die protease. Die protease-substraat mengsel is hierna deur middel van 2-D SDS-PAGE geanaliseer om te kyk of daar enige kliewing van die vier substrate voorgekom het. Alhoewel al die teiken fusieproteïene suksesvol gekloneer, uitgedruk en gesuiwer is, het die protease proewe geen kliewing getoon vir enige van die vier potensiële substrate nie. Moontlike verklarings vir hierdie negatiewe resultaat is die volgende: (1) ensiem en substrate was moontlik onsuiwer; (2) bufferkondisies was moontlik nie korrek nie; (3) gehipotiseerde substrate mag moontlik nie substrate van mycosin-3 wees nie; en (4) nie-korrekte vouing of modifisering van die teiken proteïene kon moontlik plaasgevind het. Toekomstige navorsing sal daarop gemik wees om hierdie beperkinge aan te spreek om sodoende die funksie en substrate van mycosin-3 te kan ontdek en nuwe middels teen M. tuberculosis te ontwerp.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The current anti-TB drugs have had success in decreasing the number of deaths caused by TB, however, this success is limited by the emergence of drug resistant TB strains. Therefore, a novel TB therapy that limits the development of resistance has become necessary in an attempt to effectively control TB. The anti-TB drugs directly target mycobacterial enzymes, and potentiate the development of this resistance, and have therefore provided the rationale for this study. The aim was therefore to identify host macrophage genes that affect M. tb intracellular survival. The proposed alternative anti-TB therapy potentially involves the application of RNA interference (RNAi) and RNA activation (RNAa) biological processes that will target host genes, thereby inducing an indirect bactericidal effect. We hypothesized that macrophage genes that are differentially expressed by pathogenic and non-pathogenic mycobacterial species may be important in the regulation of M. tb intracellular survival. The lipid-rich mycobacterial cell wall is implicated in the excessive clumping of the mycobacterial cells in liquid culture. In order to minimize this, Tween 80 detergent was supplemented (mycobacteriaT). However, due to substantial evidence emphasising the detrimental effects of Tween 80 on the mycobacterial cell wall, mycobacteria were also cultured without Tween 80 (mycobacteriaNT), in order to investigate if the perturbed mycobacterial cell wall induced by Tween 80 affects the transcriptional response of macrophages. We endeavoured to develop a new method to culture mycobacteria without Tween 80 that will still generate single cells. We further hypothesized that the macrophage gene expression profile induced by mycobateriaNT differs from the response induced by mycobacteriaT. Differentiated THP-1 (dTHP-1) cells were infected with pathogenic and non-pathogenic mycobacteria (for 3 h, 24 h and 48 h with M. tb and M. bovis BCG, and 3 h and 8 h with M. smegmatis) cultured in the presence or absence of Tween 80. The expression of 12 macrophage genes, selected based on their involvement in the phagocytic pathway and autophagy, as well as their general involvement in the immune response, was determined by qRT-PCR and further analysed on the REST programme. The expression of each target gene was normalised relative to the expression of the reference gene (Beta actin). We observed that out of the 12 genes, TLR7 and VAMP7 were consistently downregulated in dTHP-1 cells infected with M. tbNT and upregulated in dTHP-1 cells infected with M. smegmatisNT. Their response to M. bovis BCG was inconsistent and not significantly different, and therefore could not be interpreted. Furthermore, CCL1 was upregulated by all the mycobacterial species. However, its expression was more pronounced in response to mycobacteriaNT, when compared to mycobacteriaT. Differential gene expression of TLR7 and VAMP7 in response to pathogenic and non-pathogenic mycobacteriaNT suggests that these 2 genes may be potential targets for RNAa-based anti-TB therapy, even though we could not conclude whether their response was specific to macrophages. In addition, the observed difference in the expression of CCL1 induced by mycobacteriaNT, compared to mycobacteriaT suggests that the perturbation caused by Tween 80 on the mycobacterial cell wall most likely affected the response of macrophages to infection with mycobacteria. Furthermore, this study has demonstrated a feasible method by filtration to generate single cells from mycobacteriaNT, which should be considered for future mycobacterial infection studies. / AFRIKAANSE OPSOMMING: Die huidige anti-tuberkulose middels se sukses lê daarin dat dit die aantal sterftes verminder maar hierdie sukses word weer beperk met die ontstaan van middel-weerstandige M.tb stamme. Daarom is nuwe middels nodig wat die ontwikkeling van middel-weerstandigheid beperk in ʼn poging om effektiewe TB behandeling te bewerkstellig. Anti-tuberkulose middels teiken hoofsaaklik mycobakteriële ensiemsisteme en ontlok sodoende weerstandigheid in M.tb stamme en dit vorm die rasionale vir hierdie studie. Die doel was om gasheer makrofaag gene te identifiseer wat M.tb oorlewing intrasellulêr bewerkstellig. Die voorgestelde alternatiewe anti-TB behandeling sal dan behels die toepassing van RNA intervensie (RNAi) en RNA aktivering (RNAa) tegnologie wat gasheer selgene teiken (inaktiveer) en sodoende ʼn bakterisidiese respons induseer. Die kanse is skraal dat mycobakterieë weerstandigheid sal kan ontwikkel onder hierdie omstandighede. Ons hipotetiseer dus dat makrofaag gene wat differensieel uitgedruk word deur patogeniese en nie-patologiese mycobakteriële spesies belangrik mag wees vir die oorlewing van M.tb intrasellulêr. Die lipiedryke selwand van mycobakterieë word geïmpliseer in die oormatige sameklomping van die bakterieë in vloeistofkulture. Om hierdie effek te minimaliseer word Tween 80 normaalweg tot die medium gevoeg (mycobakterieëT). Maar weens genoegsame bewyse dat Tween-80 die selwand van bakterieë nadelig beïnvloed, is mycobakterieë ook in die afwesigheid van Tween 80 gekultureer (mycobakterieëNT) om te bepaal of die nadelige effek van Tween 80 op die selwand die transkripsionele respons in makrofage beïnvloed post-infeksie. Dit was daarom ook ons doelstelling om ʼn nuwe tegniek te ontwikkel om mycobakterieë te kultureer in die afwesigheid van Tween 80 wat ook enkelselle sal genereer vir beter gekontroleerde makrofaag infeksie. Ons hipotetiseer ook verder dat makrofaag geenuitdrukking-profiele verskil afhangende of infeksie gedoen is met mycobakterieë wat in die afwesigheid of teenwoordigheid van Tween 80 gekultureer is. Gedifferensieerde THP-1 (dTHP-1) was geïnfekteer met patogeniese en nie-patogeniese mycobakterieë (vir 3 h, 24 h en 48 h met M.tb en M.bovis BCG, en 3 h en 8 h met M.smegmatis) gekultureer in die teenwoordigheid en afwesigheid van Tween 80. Die uitdrukking van 12 makrofaag gene, geselekteer op grond van hul betrokkenheid in die fagositose meganisme en in outofagie asook hul betrokkenheid in die immuunrespons, is gekwantifiseer met qRT-PCR en daaropvolgens geanaliseer met die REST-program. Die uitdrukking van elke geen is genormaliseer relatief tot die uitdrukking van die verwysingsgeen (Beta actin). Daar is bevind dat van die 12 gene, TLR7 en VAMP7 deurlopend afgereguleer was in dTHP-1 selle geïnfekteer met M.tbNT en opgereguleer was in dTHP selle geïnfekteer met M.smegmatisNT. Selrespons met M.bovis BCG was onbeduidend en derhalwe kon geen gevolgtrekking hier gemaak word nie. Ook, CCL1 was opgereguleer met infeksie deur enige van die mycobakteriële spesies, maar CCL1 se uitdrukking was groter in respons tot mycobakterieëNT wanneer vergelyk word met respons tot mycobakterieëT. Differensiële geenuitdrukking van TLR7 en VAMP7 in respons tot patogeniese en nie-patogeniese mycobakterieëNT impliseer dat hierdie twee gene potensiële teikens kan wees vir RNAa-gebaseerde anti-TB behandeling, alhoewel ons nie kon beslis of hierdie respons spesifiek vir makrofage was nie. Ook, die verskille waargeneem in die uitdrukking van CCL1 geïnduseer deur mycobakterieëNT, vergeleke met mycobakterieëT, impliseer dat die steuring in die selwand veroorsaak deur Tween 80, heelwaarskynlik die respons van die makrofaag beïnvloed het. Hierdie studie beskryf ook ʼn filtrasiemetode om enkele mycobakteriële selle te genereer wat oorweeg moet word by toekomstige mycobakteriële infeksiestudies.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection without showing signs of illness. Steroid hormones such as glucocorticoids (GCs) however can lead to reactivation of TB. Currently two different injectable contraceptives are available in South Africa, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET). MPA is able to bind to and activate the glucocorticoid receptor (GR) and possesses selective GC activity, a pharmacological characteristic absent in NET. The aim of this study was to investigate the immune modulatory effects of the two contraceptives MPA and NET on immune responses to mycobacteria in vitro and in vivo. Human peripheral blood mononuclear cells (PBMCs) were infected with BCG (M. bovis Bacille Calmette-Guérin) and treated with MPA, NET, progesterone or cortisol and cytokine expression was measured in order to determine whether the synthetic progestins mimic endogenous progesterone or the GC cortisol. MPA, but not NET suppressed the expression of IFN-γ, IL-1α, IL-1β, IL-2, IL-12p40 and IL-13 similarly to cortisol. Furthermore expression of miRNAs, small double stranded RNA molecules that bind to complementary sequences in mRNAs of target genes and cause their degradation, was determined under the different experimental conditions. The expression of several miRNAs including miR-30c, miR-222, miR-454 and miR-331-3p were differentially influenced by MPA, cortisol and/or NET in PBMCs stimulated with BCG. For example, BCG induced the expression of miR-454 (p=0.003) which was then inhibited to basal levels by cortisol (p=0.008), MPA (p=0.002) and NET (p=0.002). These results demonstrate that steroid hormones including the contraceptives MPA and NET can modulate immune responses to mycobacteria at the miRNA level. To determine the effect of MPA and NET on BCG-induced expression of miRNAs in vivo a mouse model was used. C57BL/6 mice were injected weekly with either MPA or NET using a dose equivalent to humans and then infected with BCG. Mice treated with MPA had a higher spleen bacterial load 21 days after infection compared to both NET treated and control mice (p=0.023). In whole blood, MPA and NET treatment suppressed the BCG-induced production of miR-100 and miR-509-3p to basal levels. In contrast to NET, MPA induced expression of miR-99a expression independent of BCG infection. In the lung, the site of disease, a total number of 22 out of 377 miRNAs tested were differentially expressed 21 days after infection. The results of this study indicate that both synthetic progestins altered the immune response to BCG at the level of cytokine expression as well as the miRNA level. MPA was found to mimic cortisol by inhibiting secretion of inflammatory cytokines whereas NET appeared to have more progestogenic properties. Each of the steroid hormones was observed to induce a characteristic miRNA expression profile, both in vitro as well as in vivo, although not identical. These results highlight that the two contraceptives – although used interchangeably by women in developing countries - are pharmacologically unique and differentially modulate immune responses to mycobacteria. These data support the urgent need for further research into the link between hormonal contraceptives and susceptibility to infectious diseases. / AFRIKAANSE OPSOMMING: Individue wat latent met Mycobacterium tuberculosis ( M.tb ) geïnfekteer is, onderdruk die infeksie en wys geen simptome van die siekte nie. Steroïed hormone soos glukokortikoïede (GKe) kan egter tot die heraktivering van TB lei. Daar is tans twee verskillende inspuitbare voorbehoedmiddels beskikbaar in Suid-Afrika, medroksiprogesteroon-asetaat (MPA) en noretisteroon enantaat (NET). MPA is staat om aan die glukokortikoïed reseptor (GR) te bind en dit te aktiveer. MPA beskik ook selektiewe GK aktiwiteit, 'n farmakologiese eienskap wat afwesig is in NET. Die doel van hierdie studie was om die immuun-regulerende effekte van die twee voorbehoedmiddels, MPA en NET, op immuunresponse teen mikobakterieë in vitro en in vivo te ondersoek. Menslike perifêre bloed mononukleêre selle (PBMSe) is met BCG geïnfekteer en met MPA, NET, progesteroon of kortisol behandel. Sitokien uitdrukking was gemeet om vas te stel of die sintetiese progestiene, endogene progesteroon of die GK kortisol naboots. MPA, maar nie NET, onderdruk die produksie van IFN-γ, IL- 1α, IL- 1β, IL- 2, IL- 12p40 en IL- 13 soortgelyk aan kortisol. Verder is uitdrukking van miRNAs, klein dubbelstring RNS molekules wat aan komplimentêre volgorde in mRNAs van teiken gene bind en wat hul degradering veroorsaak, bepaal in elk van die verskillende eksperimentele toestande. Die uitdrukking van verskeie miRNAs insluitende miR-30c, miR-222, miR-454 en miR-331-3p is differensieël beïnvloed deur MPA, kortisol en/of NET in PBMSe wat met BCG gestimuleer is. Byvoorbeeld, BCG veroorsaak die uitdrukking van miR-454 wat dan geïnhibeer word tot agtergrondvlakke deur kortisol, MPA en NET. Hierdie resultate toon dat steroïed hormone, insluitend die voorbehoedmiddels MPA en NET, die immuunrespons teen mikobakterieë op miRNA-vlak affekteer. Om die effek van MPA en NET op BCG-geïnduseerde uitdrukking van miRNAs in vivo te bepaal, is ʼn muismodel gebruik. C57BL/6 muise is weekliks met 'n dosis van MPA of NET, ekwivalent aan dosisse gebruik in die mens, ingespuit en met BCG geïnfekteer. Muise wat met MPA behandel is, het 'n hoër bakteriële lading in die milt 21 dae na infeksie in vergelyking met NET-behandelde muise en kontrole muise. In hul bloed, onderdruk MPA en NET behandeling die BCG-geïnduseerde produksie van miR-100 en miR-509-3p tot basale vlakke. In teenstelling met NET, induseer MPA die uitdrukking van miR-99a onafhanklik van BCG infeksie. In die long is 'n totaal van 23 miRNAs differensieël uitgedruk 21 dae na die infeksie. Die resultate van hierdie studie dui daarop dat beide sintetiese progestien die immuunrespons teen BCG verander op sitokien sowel as miRNA vlak. MPA boots hoofsaaklik kortisol na deur inhibering van sitokien-produksie terwyl NET meer progesterone eienskappe het. Op miRNA vlak veroorsaak elk van die steroïed hormone 'n kenmerkende miRNA uitdrukkingsprofiel, beide in vitro asook in vivo. Hierdie resultate beklemtoon dat die twee voorbehoedmiddels - alhoewel hulle afwisselend gebruik word deur vroue in ontwikkelende lande - farmakologies uniek is en differensieël die immuunrespons reguleer teen Mycobacterium. Hierdie data ondersteun die dringende behoefte aan verdere navorsing in verband met hormonale voorbehoedmiddels en vatbaarheid vir aansteeklike siektes.

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Central dogma suggests that mutations in target genes is the primary cause of resistance to first and second-line anti-TB drugs in Mycobacterium tuberculosis. However, it was previously reported that approximately 5% of Rifampicin mono-resistant clinical M. tuberculosis did not harbor mutations in the rpoB gene. The present study hypothesized that active efflux plays a contributory role in the level of intrinsic resistance to different anti-TB drugs (Isoniazid, Ethionamide, Pyrazinamide, Ethambutol, Ofloxacin, Moxifloxacin, Ciprofloxacin, Streptomycin, Amikacin and Capreomycin in RIF mono-resistant clinical M. tuberculosis isolates with a rpoB531 (Ser-Leu) mutation. This study aimed to define the role of Efflux pump inhibitors (verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine) in enhancing the susceptibility to different anti-TB drugs in the RIF mono-resistant clinical isolates. The isolates were characterized by determining the level of intrinsic resistance to structurally related/unrelated anti-TB drugs; determining the effect of EPIs on the level of intrinsic resistance in the isolates and comparing the synergistic properties of the combination of EPIs and anti-TB drugs. To achieve this, genetic characterization was done by PCR and DNA sequencing. Phenotyping was done by the MGIT 960 system EpiCenter software to determine the MICs of the different anti-TB drugs and the effect of verapamil and carbonylcyanide m-chlorophenylhydrazone on determined MICs. Due to inability to test reserpine in a MGIT, a different technique (broth microdilution) was used for the reserpine experiment. Additionally; fractional inhibitory concentrations (FIC) indices were calculated for each of these drugs. The FIC assess the anti-TB drugs/inhibitor interactions. STATISTICA Software: version 11 was used for statistical analysis. Results revealed that the RIF mono-resistant isolates were sensitive at the critical concentrations of all 10 drugs tested, with the exception of Pyrazinamide. This could be explained by the technical challenges of phenotypic Pyrazinamide testing. A significant growth inhibitory effect was observed between the combination of EPI and anti-TB drug exposure in vitro. This suggests that verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine play a significant role in restoring the susceptibility (decrease in intrinsic resistance level) of the RIF mono-resistant isolates to all anti-TB drugs under investigation. Additionally, a synergistic effect was observed by the combination treatment of the anti-TB drugs with the different EPIs. Based on these findings, we proposed a model suggesting that efflux pumps are activated by the presence of anti-TB drugs. The activated pumps extrude multiple or specific anti-TB drugs out of the cell, this in turn decrease the intracellular drug concentration, thereby causing resistance to various anti-TB drugs. In contrast, the addition of EPIs inhibits efflux pump activity, leading to an increase in the intracellular drug concentration and ultimate cell death. This is the first study to investigate the effect of different efflux pumps inhibitors on the level of intrinsic resistance to a broad spectrum of anti-TB drugs in drug resistant M. tuberculosis clinical isolates from different genetic backgrounds. The findings are of clinical significance as the combination of treatment with EPI and anti-TB drugs or use of EPIs as adjunctives could improve MDR-TB therapy outcome. / AFRIKAANSE OPSOMMING: Sentrale dogma beweer dat mutasies in teiken gene die primêre oorsaak van die weerstandheid teen anti-TB-middels in Mycobacterium tuberculosis is. Vorige studies het getoon dat ongeveer 5% van Rifampisien enkelweerstandige kliniese M. tuberculosis isolate nie ‘n mutasie in die rpoB geen het nie. Die hipotese van die huidige studie was dat aktiewe pompe 'n bydraende rol speel in die vlak van intrinsieke weerstandheid teen 10 verskillende anti-TB-middels (Isoniasied, Ethionamied, Pyrazinamied, Ethambutol, Ofloxacin, Moxifloxacin, Siprofloksasien, Streptomisien, Amikasien and Capreomycin) in RIF enkelweerstandige kliniese M . tuberculosis isolate met 'n rpoB531 (Ser-Leu) mutasie. Die doel van hierdie studie was om die rol van uitpomp inhibeerders (verapamil, carbonylcyanide m-chlorophenylhydrazone en reserpien) te definieer in die verbetering van die werking vir verskillende anti-TB-middels in die RIF enkelweerstandige kliniese isolate. Die doelstellings van die studie was om die vlak van intrinsieke weerstandigheid teen struktureel verwante/onverwante anti-tuberkulose middels asook die effek van die EPIs op die vlak van intrinsieke weerstand in die isolate is bepaal. Verder is sinergistiese eienskappe van die kombinasie van EPIs en anti-TB-middels ondersoek. Hierdie doelstellings is bereik deur genetiese karakterisering deur PKR en DNS volgorde bepaling. Fenotipering is gedoen deur gebruik te maak van MGIT 960 EpiCenter sagteware om die Minimum Inhibisie Konsentrasie (MIC) van die verskillende anti-TB-middels en die effek van verapamil en carbonylcyanide m-chlorophenylhydrazone op die MIC te bepaal. Reserpien kan nie in die MGIT sisteem getoets word nie, and daarom is 'n ander tegniek (mikro-verdunning) is gebruik om die effek van reserpien te toets. Fraksionele inhiberende konsentrasies (FIC) is bereken vir elk van hierdie middels die anti-TB-middels / inhibeerder interaksies te bepaal. STATISTICA v11 sagteware is gebruik vir alle statistiese analises. Resultate van hierdie studie toon dat die RIF enkelweerstandige isolate sensitief is teen kritieke konsentrasies van al die middels, met die uitsondering van Pyrazinamied. Weerstandigheid van Pyrazinamied kan wees as gevolg van welbekende tegniese probleme met die standaard fenotipiese pyrazinamied toets. ‘n Beduidende groei inhiberende effek is waargeneem tussen die kombinasie van EPI en anti-TB middel blootstelling in vitro. Dit dui daarop dat verapamil, CCCP en reserpine 'n belangrike rol speel in die herstel van die sensitiwiteit (afname in intrinsieke weerstand vlak) van die RIF enkelweerstandige isolate aan alle anti-TB-middels wat ondersoek is. Daarbenewens is 'n sinergistiese effek waargeneem deur die kombinasie van die verskillende anti-TB-middels en die verskillende EPIs. Op grond van hierdie bevindinge het ons ‘n model voorgestel wat toon dat uitvloei pompe geaktiveer word deur die teenwoordigheid van anti-TB-middels en die geaktiveerde pompe dan verskeie of spesifieke anti-TB-middels uit die sel pomp. Dus verminder die intrasellulêre konsentrasie van die middel en veroorsaak daardeur weerstandigheid teen verskeie anti-TB-middels. Die byvoeging van EPIs inhibeer uitvloei pompe se werking en lei tot 'n toename in die intrasellulêre konsentrasie van die middels en uiteindelik die dood van die selle. Hierdie is die eerste studie wat die effek van verskillende uitvloei pompe inhibeerders op die vlak van intrinsieke weerstand teen 'n breë spektrum van anti-TB-middels in die middel-weerstandige kliniese isolate ondersoek. Die bevindinge kan van belangrike kliniese belang wees aangesien die kombinasie van behandeling met EPI en anti-TB-middels die uitkoms MDR-TB terapie kan verbeter.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Tuberculosis (TB) is a major cause of morbidity and mortality across the world, affecting adults and children. Children infected with TB differ from adults, as their immunological and patho-physiological response to the disease is different. Although there are a variety of tests available for TB diagnosis, they have limitations when used to diagnose paediatric TB. Children are also unable to generate sputum spontaneously when required for the use in culture or microscopy as diagnostic method. Children however do produce sputum, containing the TB bacilli, which they swallow. If the TB bacilli can therefore be retrieved from the stomach and tested, TB can be diagnosed using gastric samples. In this thesis, a variety of styrene maleimide copolymer (SMI) derivatives were prepared as potential M. tuberculosis-capturing platforms. This was done by modifying poly(styrene-co-maleic anhydride) (SMA) with a variety of primary amine compounds, selected based on possible chemical interactions with the M. tuberculosis cell wall. All the prepared copolymer derivatives were electrospun into nanofibrous mats using the single needle electrospinning technique to yield SMI nanofibers, functionalized with different compounds. Some of the functionalized SMI nanofibers were prepared by surface-functionalization of the polymer nanofibers after electrospinning and some by modification of the polymer before electrospinning. Affinity studies were conducted at neutral and low pH between the different functionalized SMI nanofibers and two mycobacterium strains, namely the bacillus Calmette-Guérin strain of Mycobacterium bovis (BCG) and M. tuberculosis, to evaluate the surfaces of the modified SMI nanofibers as mycobacterium-capturing platforms. The successful capture of BCG onto the surfaces of the various functionalized nanofibers was confirmed by SEM and fluorescence microscopy (FM). Analysis of the SEM and FM images indicated that the SMI nanofibers, functionalized with a C12 aliphatic quaternary ammonium moiety (SMI-qC12), captured BCG the most effectively through a combination of ionic and hydrophobic interaction. Concentration and time studies revealed that the extent of this interaction was dependent on incubation time and concentration of BCG. The affinity studies with BCG also concluded that the polymer used for the nanofibrous-capturing platform should not be too hydrophobic in character as this caused poor wetting of the functionalized nanofibers, thus preventing close contact with the mycobacteria and a reduction in the capture effectivity of the polymer nanofibers. The successful capture of M. tuberculosis onto the SMI-qC12 nanofibrous surface was confirmed by FM, light microscopy (LM) and polymerase chain reaction (PCR). The extent of this interaction was dependent on the concentration of M. tuberculosis. The detection of M. tuberculosis using FM and LM as detection methods was simplified by the tendency of M. tuberculosis to clump together in clusters on the hydrophobic surface of the SMI-qC12 nanofibers. As a result of this clustering, FM and LM were therefore regarded as feasible detection methods to image M. tuberculosis on the surface of the SMI-qC12 nanofibers, even at relatively low concentration of M. tuberculosis. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is 'n groot oorsaak van morbiditeit en mortaliteit regoor die wêreld en affekteer volwassenes en kinders. Kinders wat met TB geïnfekteer is, se immunologiese en patofisiologiese reaksie op die siekte verskil van die van volwassenes en dit het belangrike implikasies vir die diagnose van TB in kinders. Alhoewel daar 'n verskeidenheid van toetse beskikbaar is vir die diagnose van TB, het hulle beperkings wanneer dit gebruik word om pediatriese TB te diagnoseer. Kinders kan ook nie spontaan sputum produseer as dit nodig is vir die gebruik in kultuur of mikroskopie as diagnostiese metode. Kinders produseer egter wel sputum, wat die TB basille bevat, wat hulle dan insluk. As die TB basille uit die maag versamel kan word en getoets kan word, kan TB gediagnoseer word met behulp van maag monsters. In hierdie tesis is 'n verskeidenheid van stireen maleimied kopolimeer (SMI) afgeleides voorberei as potensiële Mycobacterium tuberkulose (Mtb)-vaslegging platforms. Dit is gedoen deur die modifikasie van poli(stireen-ko-maleïen anhidried) (SMA) met 'n verskeidenheid primêre amien verbindings as oppervlak-funksionaliseringsagente. Hierdie primêre amien verbindings is gekies op grond van moontlike chemiese interaksies met die Mtb selwand. Al die voorbereide kopolimeer afgeleides is elektrogespin in nanoveselagtige matte met behulp van die enkel-naald elektrospin tegniek om SMI nanovesels te lewer wat gefunksionaliseer is met verskillende verbindings. Sommige van die gefunksionaliseerde SMI nanovesels is berei deur oppervlak-funksionalisering van die polimeer nanovesels na elektrospin, en sommige deur die modifikasie van die polimeer voor elektrospin. Affiniteitstudies is uitgevoer, by neutrale en lae pH, tussen die verskillende gefunksionaliseerde SMI nanovesels en twee mikobakterium rasse, naamlik die basillus Calmette-Guérin ras van Mycobacterium bovis (BCG) en M. tuberculosis, om die oppervlaktes van die gewysigde SMI nanovesels te evalueer as mikobakterium-vaslegging platforms. Ontleding van die SEM en FM beelde het aangedui dat die SMI nanovesels, gefunksionaliseer met 'n C12 alifatiese kwaternêre ammonium groep (SMI-qC12), BCG die doeltreffendste vasgevang het deur 'n kombinasie van ioniese en hidrofobiese interaksie. Konsentrasie- en tydstudies tussen BCG en SMI-qC12 het aangedui dat die omvang van hierdie interaksie afhanklik is van inkubasietyd en konsentrasie van BCG. Die affiniteitstudies met BCG het ook aangedui dat die polimeer wat gebruik word vir die nanoveselagtige-vaslegging platform nie te hidrofobiese moet wees nie, aangesien dit swak benatting van die gefunksionaliseerde nanovesels veroorsaak, en dus noue kontak met die mikobakterieë voorkom met ʼn gevolglike vermindering in die vasvang-effektiwiteit van die polimeer nanovesels. Die suksesvolle vasvang van M. tuberculosis op die SMI-qC12 nanovesels is bevestig deur FM, lig mikroskopie (LM) en polimerase kettingreaksie (PKR). Die opsporing van Mtb deur die gebruik van FM en LM as opsporingmetodes is vergemaklik deur die tendens van Mtb om in groepies saam te pak op die hidrofobiese oppervlak van die SMI-qC12 nanovesels. As gevolg van hierdie groepering, is FM en LM dus haalbare opsporingmetodes om M. tuberculosis op die oppervlak van die SMI-qC12 nanovesels waar te neem, selfs by relatief lae konsentrasie van M. tuberculosis.

Thesis (DMed (Medicine. Internal Medicine))-University of Stellenbosch, 2005. / Mycobacterium tuberculosis (M. tuberculosis) accounts for more adult deaths than any other infectious agents. The present study included 162 patients with tuberculous pericarditis; 50% of the tuberculous pericarditis patients studied were human immunodeficiency virus (HIV) positive, compared to only 4.2% of patients who presented with non-tuberculous pericardial effusions. A steady year-to-year rise in HIV prevalence was observed in this 6-year study. Although the prognosis of pericardial tuberculosis (TB) is excellent with appropriate medical treatment, untreated pericardial TB has a mortality of 80-85%. It is thus important to diagnose tuberculous pericarditis efficiently. Traditionally, the diagnosis of pericardial TB is established by positive mycobacterial culture and/or histological evidence of necrotising granulomatous inflammation of the pericardium. Our study confirmed the insensitivity of pericardial fluid culture and pericardial biopsy in the diagnosis of pericardial TB, and at the time of clinical decision-making, results were usually not available. To overcome these difficulties, we explored various alternative strategies and this resulted in two diagnostic tools, namely a diagnostic rule and a diagnostic algorithm or classification tree. By means of classification and regression tree analysis, we allocated a weighted diagnostic index to each of five independently predictive features (fever, night sweats, weight loss, serum globulin >40 g/L and peripheral blood leukocyte count <10x109/L). A total diagnostic index of 6 or more corresponded to 82-86% sensitivity and 76-87% specificity for a diagnosis of tuberculous pericarditis. When possible, pericardial fluid should be aspirated to determine adenosine deaminase (ADA) levels and pericardial differential leukocyte counts. Fluid should also be sent for Gram stain and culture. The proposed diagnostic classification tree utilises the independently predictive attributes of pericardial adenosine deaminase levels, pericardial fluid lymphocyte/neutrophil ratios, peripheral leukocyte counts and the HIV status. Applying this prediction model to our entire data set of 233 patients resulted in 96% sensitivity and 97% specificity for the correct diagnosis of tuberculous pericarditis. Generally, patients were critically ill at the time of enrolment; 90% of tuberculous pericarditis presented with echocardiographic features of cardiac tamponade. Echoguided percutaneous pericardiocentesis with an indwelling catheter and intermittent daily aspiration was highly effective and safe. It is likely that the combination of this drainage technique and the early initiation of anti-tuberculous chemotherapy contributed to the almost complete absence of constriction in the patients studied, and our data do not support the routine use of adjunctive corticosteroids in patients with tuberculous pericarditis. Tuberculous exudates result from a Th1 mediated immune response characterised by lymphocyte dominance, significantly elevated levels of gamma-interferon (IFN-γ) and undetectable levels of interleukin-4 (IL-4). IFN-γ levels were not influenced by HIV status in spite of the severely diminished pericardial CD4+ lymphocyte counts observed in this study. It is thus likely that in HIV positive patients IFN-γ production is partly maintained by activated CD8+ T cells, which were significantly elevated in HIV positive patients compared to HIV negative tuberculous pericarditis patients. This finding underlines the importance of IFN-γ in the human immune response against M. tuberculosis. We also demonstrated that the presence of ADA in pericardial fluids reflects the activity of the cellular immune response. Both IFN-γ and ADA can be utilised as sensitive and specific diagnostic tools for pericardial TB.

Thesis (MSc)--Stellenbosch University, 2016. / ENGLISH ABSTRACT: Tuberculosis (TB) is one of the world’s deadliest diseases, with one third of the population being infected by it. The diagnosis of active tuberculosis entails finding and identifying Mycobacterium tuberculosis (Mtb), the causative pathogen in a specimen of bodily fluid from the patient. Multiple samples will improve the diagnostic yield and specimen volumes should therefore be as large as possible, which is often challenging for patients and especially younger children. Alternatively, a smaller volume could be required if there was a manner in which to concentrate the bacteria within a specimen, through use of a substrate which has an affinity for the pathogenic species. Polymers having intrinsic cellular activity are of interest as such substrates, one such being the natural polysaccharide, chitosan. In this thesis, a variety of modified chitosan derivatives were prepared as potential Mtb-capturing substrates. This was achieved by modifying chitosan with a variety of moieties, selected based on possible interactions with the Mtb cell wall, to render various quaternary ammonium salts of the polymer chitosan. The quaternized chitosan derivatives were then used to synthesize nano-substrates having an affinity for Mtb. Polymer coated superparamagnetic magnetite nanoparticles (SPMNs) were synthesized via an in situ co-precipitation technique, in which modified chitosan is able to chelate with the metal core. Polymer nanofibers were also electrospun via the electrospinning technique. The prepared derivative, N-trimethylammonium chitosan chloride (TMC), was electrospun into nanofibers by blending with suitable non-ionogenic polymers, namely polyvinyl alcohol (PVA), polyethylene oxide (PEO), polyvinyl pyrrolidone (PVP) and polyacrylamide (PAM), required to facilitate nanofiber formation. Affinity studies were conducted between the modified chitosan nano-substrates and the bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis, the attenuated Mtb-mimic bacteria, for evaluation as mycobacterium capturing substrates. The successful capture of BCG onto the surfaces of the various modified chitosan nanofibers and modified chitosan coated superparamagnetic nanoparticles was confirmed by fluorescence microscopy (FM), light microscopy (LM), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM). Analysis of the FM, TEM and FE-SEM images indicated that the chitosan coated nanoparticles functionalized with a C12 aliphatic quaternary ammonium moiety (CS-qC12), captured the most BCG through a combination of ionic and hydrophobic interaction. TMC blended with PVA, to produce nanofibers crosslinked with genipin, were found to have the strongest interaction with BCG of the nanofibrous mats tested. These findings were corroborated by water contact angle measurements, which established that PVA was the least hydrophilic of the non-ionogenic polymers and had hydrogen bond donating groups only, factors influencing the cellular adhesive properties of affinity substrates. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is een van die wêreld se mees dodelikste siektes, met ‘n derde van die bevolking wat geïnfekteer is daarmee. Ten einde aktiewe TB te diagnoseer moet Mycobacterium tuberculosis (Mtb), die voorsakende patogeen in ʼn monster van die pasiënt se liggaamlike vloeistof, gevind en ïdentifiseer word. Veelvuldige monsters sal die diagnotiese opbrengs verhoog en monster volumes moet dus so groot as moontlik wees wat dikwels ʼn uitdaging vir pasiënte en veral jonger kinders kan bied. Alternatiewelik kan ʼn kleiner monster van die pasiënt vereis word indien daar ʼn manier was om die bakterieë in ʼn monster te konsentreer deur die gebruik van ʼn substraat wat ʼn affiniteit toon vir die patogeniese spesie. Polimere met ʼn intrinsieke sellulêre aktiwiteit, wek belangstelling as sodanige substraat, een synde die natuurlike polisakkaried, chitosan. In hierdie tesis is ʼn verskeidenheid gemodifiseerde chitosan afgeleides voorberei as potensiële Mtb-vaslegging substrate. Dit is gedoen deur chitosan te modifiseer met ʼn verskeidenheid funksionele groepe, gekies op grond van moontlike interaksies met die Mtb selwand, ten einde ʼn verskeidenheid kwaternêre ammonium soute van die chitosan polimeer te bekom. Die kwaternêre chitosan afgeleides is gevolglik gebruik om nano-substrate te sintetiseer wat ʼn affiniteit toon vir Mtb. Polimeer bedekte superparamagnetiese magnetiet nanopartikels (SPMNs) is gesintetiseer via ʼn in situ mede-neerslag metode, waarvolgens die gemodifiseerde chitosan polimere in staat is om met die metaal kern te chelaat. Polimeer nanovesels is ook geëlektrospin deur die elektrospin tegniek te gebruik. Die voorbereide afgeleide N-trimetielammonium chitosan chloried (TMC) is tot nanovesels geëlektrospin deur vermenging met geskikte nie-ionogeniese polimere, naamlik poliviniel-alkohol (PVA), polietilene-oksied (PEO), poliviniel-pirrolidoon (PVP) en poliakrielamied (PAM), wat vereis word ten einde nanovesels te produseer. Affiniteit studies is uitgevoer tussen die gemodifiseerde chitosan nano-substrate en die bacillus Calmette-Guérin (BCG) stam van Mycobacterium bovis, die verswakte Mtb-mimiek bakterieë vir evaluering as mycobakterium-vaslegging substrate. Die suksesvolle vasvang van BCG op die oppervlaktes van die verskillende gemodifiseerde chitosan nanovesels en gemodifiseerde chitosan bedekte SPMNs is bevestig deur fluoressensie mikroskopie (FM), lig mikroskopie (LM), transmissie elektron mikroskopie (TEM) en veld-emissie-skandering elektron mikroskopie (FE-SEM). Analise van die FM, TEM en FE-SEM beelde het getoon dat die chitosan bedekte nanopartikels met byvoeging van ʼn C12 alifatiese kwaternêre ammonium groep, die meeste BCG vasgevang het deur ʼn kombinasie van ioniese en hidrofobiese interaksie. TMC vermeng met PVA om nanovesels te vorm, gekruisbind met genipin, is gevind om die sterkste interaksie met BCG te toon. Hierdie bevindings is bevestig deur water-kontak-hoek-metings, wat getoon het dat PVA die minste hidrofilies van die nie-ionogeniese polimere was en slegs waterstof-binding skenkings groepe het, alles faktore wat die sellulêre bindingskwaliteite van affiniteit-substrate sal beïnvloed.