Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The immune system matures progressively from infancy to adulthood, thus children may differ from adults in their immune function. The immature immune system demonstrates a higher naive to memory T cell ratio, defective macrophage function and antigen presentation which, cumulatively, results in diminished production of cytokines such as IFN-y. This cytokine has been shown to play a pivotal role in protection against Mycobacterium tuberculosis (M. tuberculosis) disease. Other cytokines, such as IL-12 and TNF-a, are also involved in the defence against M. tuberculosis. Epidemiological evidence suggests an agerelated incidence of tuberculosis (TB) irrespective of prevalence in a given region. Reports in the literature also demonstrate depressed immune responses in TB patients, at diagnosis, (before TB therapy) with subsequent improvement after TB therapy. The aims of this study were to optimise a whole blood assay in order to characterise immune responses, as measured by proliferation and cytokine production, in TB patients (after TB therapy) and healthy controls of different ages. Immune responses of TB patients would also be compared, before, and after TB therapy. A total of 68 subjects were included in this study. These comprised 27 TB patients and 41 healthy Mantoux positive controls. All subjects were stratified into two age groups: <12 years and >12 years. Diluted whole blood was cultured and stimulated with the mitogen, phytohaemagglutinin (PHA) and the specific mycobacterial antigen, purified protein derivative (PPD) to measure proliferation and IFN-y, IL-2, TNF-a and IL-10 production in the supernatant of cultures. Age was a significant variable for the following PHA-stimulated cytokines: IFN-y, TNF-a and IL-10. Proliferation and IL-2 production after PHA stimulation did not demonstrate any relationship with age. None of the PPD-stimulated proliferative or cytokine responses demonstrated any correlation with age. Concentrations of PHA- and PPD-induced IFN-y for all subjects (patients and controls) were increased “after therapy”, compared to “before therapy”. This phenomenon could possibly be due to maturation in the capacity of the immune system to produce this cytokine. Patients >12yrs demonstrated improvement in all proliferative and cytokine responses (except for PPD-induced IL-2 and TNF-a) “after therapy”, compared to “before therapy”. This is probably a valid finding and is thus in accordance with the literature. The whole blood assay is a simple, non-laborious assay that, according to the literature, produces results that seem to correlate well with that of conventionally used PBMCs. Age appears to be an important variable in the quantitative assessment of cellular immune responses (when the mitogen, PHA is used as a stimulant) and immune responses of older TB patients appear to improve after TB therapy, compared to before TB therapy. / AFRIKAANSE OPSOMMING: Die immuunsisteem matureer stelselmatig van kind na volwassene. Dus sal kinders se immuniteit verskil van volwassenes s’n. Die immature immuunsisteem het ‘n hoer nai'witeit vir geheue T-sel verhouding, defektiewe makrofaag funksie en antigeen presentering wat gesamentlik lei tot verminderde produksie van sitokiene soos byvoorbeeld IFN-y. Daar is bewys dat hierdie sitokien ‘n deurslaggewende rol speel in die beskerming teen Mycobacterium tuberculosis (M. tuberculosis). Ander sitokiene, soos IL-12 en TNF-a speel ook ‘n rol in die beskerming teen M. tuberculosis. Epidemiologiese data dui aan dat daar ‘n ouderdomverwante insidensie van tuberkulose (TB) is sonder dat dit beinvloed word deur die voorkoms van TB in ‘n sekere area. Verslae in die literatuur wys ook op onderdrukte immuniteitrespons in TB-pasiente by diagnose (voor TB-behandeling) met uiteindelike verbetering na TB-behandeling. Die doel van hierdie studie was om ’n volbloed metode te optimaliseer in ’n poging om die immuunrespons te karakteriseer soos gemeet met behulp van proliferasie en sitokien produksie by TB-pasiente (na TB-behandeling) en gesonde kontrole persone van verskillende ouderdomme. Die immuunrespons van TB-pasiente word ook vergelyk voor en na TBbehandeling. ‘n Totaal van 68 gevalle is vir die studie gebruik. Dit sluit in 27 TB-pasiente en 41 gesonde Mantoux positiewe kontroles. A1 die gevalle is in twee ouderdomsgroepe verdeel: <12 jaar en >12 jaar. Kulture is gemaak van verdunde volbloed en gestimuleer met phytohaemaglutinin (PHA) en gesuiwerde proteien derivaat (purified protein derivative-PPD) om proliferasie en IFN-y, IL- 2, TNF-a en IL-10- produksie in die supernatant van die kulture te meet. Ouderdom was ‘n beduidende veranderlike vir die volgende PHA-gestimuleerde sitokiene: IFN-y, TNF- a en IL-10. Daar was geen korrelasie tussen proliferasie en IL-2-produksie na PHA-stimulasie aan die een kant en ouderdom aan die ander kant nie. Geen van die PPDgestimuleerde proliferasie response of sitokien response het enige korrelasie met ouderdom getoon nie. Konsentrasies van PHA- en PPD-geinduseerde IFN-y vir alle gevalle (pasiente en kontrole) was verhoog “na behandeling”, vergeleke met “voor behandeling”. Hierdie fenomeen kan moontlik toegeskryf word aan maturasie in die vermoe van die immuunsisteem om sitokiene te vervaardig. Pasiente >12 jaar het bewyse getoon van verbetering in alle proliferasie en sitokien response (behalwe vir PPD-gei'nduseerde IL-2 en TNF-a) “na behandeling”, vergeleke met “voor behandeling”. Dit is waarskynlik ‘n geldige bevinding en is dus in ooreenstemming met verslae in die literatuur. Die volbloed metode is ‘n eenvoudige metode wat nie baie arbeidsintensief is nie, wat volgens die literatuur, resultate lewer wat goed korreleer met die konvensionele gebruik van perifere bloed mononukliere selle (PBMC’s). Dit wil voorkom asof ouderdom ‘n belangrike veranderlike is in die kwantitatiewe beoordeling van sellulere immuunrespons (wanneer PHA gebruik word as ‘n stimulant), en of die immuunrespons van ouer TB-pasiente verbeter na TB-behandeling in vergeleke met die respons voor TB-behandeling.

Thesis (PhD)--Stellenbosch University, 2004 / ENGLISH ABSTRACT: It is estimated that about one third of the world's population is infected with Mycobacterium tuberculosis (M. tuberculosis). Of those infected, only 10 % will develop disease of which 3-5 % will relapse after completion of treatment. Susceptibility to M tuberculosis or relapse following treatment, may be due to environmental influences such as poverty-related factors including intestinal parasites (helminths), and/or genetic factors, all of which can influence the immune response to M. tuberculosis. In the current study, the epidemiology of mycobacterial infection and helminths was studied in two adjacent suburbs of Cape Town, South Africa. These communities had a tuberculosis notification rate of over 1 000/100 000 population with rampant infestations by helminths such as Ascaris lumbricoides and Trichuris trichiura. M. tuberculosis infection and Bacille Calmette Guerin (BCG) vaccination induce a Thl (type 1) immune response, while a Th2 (type 2) immune response is required for expulsion of intestinal parasites. Type 1 and type 2 responses negatively cross regulate each other in vitro and in experimental models. The interaction of these two immune responses in the study community, were investigated. It was hypothesised that susceptibility to M. tuberculosis and progression to disease may be increased in individuals mounting prominent type 2 immune responses, manifested by high serum IgE levels. Furthermore it is proposed that that poverty-related factors and intestinal parasites, specifically those trafficking through the lungs, could further augment the type 2 dominance in the study community. Results presented show that serum IgE concentrations, surrogate marker for type 2 activation, were high among healthy adults, confirming the dominance of type 2 responses. When characterised in census blocks or enumerator sub-districts (ESDs), IgE levels correlated with the tuberculosis notification rate per ESD. The notification rate of tuberculosis also correlated with the socio-economic status, female literacy and population density of the study population. Although these correlations do not necessarily imply a causal relationship, these factors are associated with susceptibility to M. tuberculosis. It was also shown that IgE concentrations decreased significantly after successful treatment of tuberculosis, showing that IgE concentrations in humans can be down-regulated under these circumstances, presumably due to enhancement of a type 1 response. Furthermore, as a reason for the high serum IgE concentrations in the study population, the helminth burden was subsequently measured in all primary school children in the study community. Results show that more than 50 % of the children recruited were infected with A. lumbricoides and/or T. trichiura. Schools situated in the poorest areas with the highest tuberculosis notification rates, presented with the highest prevalences of helminths. All the children, irrespective of their helminth status or their participation in the study, subsequently received ant-helminthic treatment. The BCG vaccination scar status and Mantoux skin test responses were available on a sub-sample of the above-mentioned school children. Although it is assumed that most children receive BCG vaccination in the neonatal period, only two thirds of the children had evidence of a BCG scar. The results show that the prevalence of BCG scar positivity, while independent of age, was lower in children around 11 years of age. In contrast to the broad constancy of BCG scar expression, the percentage of children showing Mantoux reactivity increased with age, from 13 % at 6 years to 65 % at mid teenage. The time course of Mantoux conversion with age indicated that any tuberculin sensitivity, induced by the BCG, waned within the first few years of life and that PPD responsiveness thereafter was induced by environmental exposure to M. tuberculosis. Contrary to the ThllTh2 paradigm, the prevalence of helminth infection in children with a BCG scar was marginally lower than in those without one. A relatively weak positive correlation was found between tuberculin responsiveness and helminth infection and this correlation was most marked in children without a BCG scar. In this subgroup, children who were infected with helminths were more likely to be PPD responsive than those who were not infected. The data showed that conversion to PPD sensitivity predisposed to helminth infection. The results suggest that the effect of helminth infection on the development of clinical tuberculosis is such that those with large worm burdens and who make good PPD responses are likely to be resistant whereas those who deal very effectively with these parasites and who make weaker PPD responses are more likely to be susceptible. The data also indicate that the BCG vaccine used in this study does not give rise to a latent infection whereas the pathogenic M. tuberculosis does so and repeatedly stimulates an immune response to it. In a separate study, it was demonstrated how the host response to M. tuberculosis differs in patients at risk for developing tuberculosis after successful completion of treatment, compared to those who have protective immunity. Individuals participating in the study were also interviewed to understand their social and economic background and how it relates to the disease. Purified protein derivative (PPD) and M. tuberculosis-induced cytokine responses were determined in the study groups. The results show that single immunological marker of susceptibility could not be distinguished, but rather immunological patterns of susceptibility were observéd. Individuals who have had tuberculosis once before and who had been cured, presented with an immuno-suppressive profile, which included high concentrations of IL-lO, TGF-13 as well as high IgE levels. This type of profile suggests that although these individuals have had tuberculosis once before, they have not acquired protective immunity and would be susceptible to reinfection and progression to disease. Furthermore, the interviews conducted showed that most of the people included in this study were poor, unemployed, undernourished and lived in overcrowded conditions. It seems inevitable that those individuals with the immuno-suppressed profile living in poverty would present with a second episode of tuberculosis in the near future. We conclude that in the study community, which has a typical third world setting, poverty-related factors including helminths, could contribute to a dominant type 2 immune response which in tum, would down-regulate the protective type I response, resulting in an enhanced susceptibility to M. tuberculosis and progression to disease. / AFRIKAANSE OPSOMMING: Dit word beraam dat ongeveer een derde van die wêreld se populasie geïnfekteer is met Mycobacterium tuberculosis (M. tuberculosis). Van diegene wat wel geïnfekteer is, sal slegs 10 % siekte ontwikkel met 3-5 % wat 'n relaps episode sal ervaar na voltooiing van behandeling. Vatbaarheid vir M. tuberculosis of 'n relaps episode gevolg na behandeling, mag toegeken word aan armoede-verwante faktore wat intestinale parasiete (helminte) asook genetiese faktore, insluit. Hierdie faktore het die vermoë om die immuun respons teen M. tuberculosis te beïnvloed. In die huidige studie, is die epidemiologie van die mikobakteriele infeksie en helminte bestudeer in twee aangrensende voorstede van Kaapstad, Suid Afrika. Hierdie gemeenskappe het 'n tuberkulose aanmeldings koers van 1 000/1 00 000 populasie met verpreide infestasies met helminte soos Ascaris lumbricoides and Trichuris trichiura. Infeksie met M tuberculosis en vaksinasie met Bacille Calmette Guerin (BeG), induseer 'n Th1 (tipe 1) immuun respons, terwyl 'n Th2 immuun respons benodig word vir die eliminasie van intestinale parasiete. Die interaksie tussen die twee immuun response was in die huidige studie populasie bestudeer. Dit word gepostuleer dat persone met 'n sterk tipe 2 immuun respons, gemanifesteer deur hoë serum IgE vlakke, vatbaar is vir infeksie met M. tuberculosis en progressie tot siekte. Verder was dit voorgestel dat armoede-verwante faktore en intestinal parasiete, veral parasiete wat deur die longe beweeg, 'n dominante tipe 2 respons verder kan versterk. Die resultate voorgestel, wys daarop dat serum IgE konsentrasies, 'n surrogaat merker vir tipe 2 aktivering, hoog was in gesonde volwassenenes. Dit het die siening van 'n dominante tipe 2 respons bevestig. IgE vlakke was bereken vir elke sensus blok of enumerator sub-distrik (ESD) en het gekorreleer met die tuberkulose annmeldings koers per ESD. Die aanmeldings koers het ook gekorreleer met die sosio-ekonomiese status, vroulike geletterdheid en populasie digtheid. Alhoewel hierdie korrelasies nie noodwending dui op 'n oorsaak en gevolg verhouding nie, is dit duidelik dat hierdie faktore kan bydra tot vatbaarheid vir M. tuberculosis. Dit was ook getoon dat IgE konsentrasies beduidend afneem na suksesvolle behandeling van tuberkulose. Dit wys daarop dat IgE konsentrasies in mense afgeruleer kan an waarskynlik dui op 'n verhoogde tipe 1 respons. Verder, as 'n rede vir die hoë IgE konsentrasies in die studie populasie, is die helmint ladings gevolglik in alle prim ere skool kinders in die studie populasie, gemeet. Die resultate dui daarop dat meer as 50 % van die kinders ingesluit, geïnfekteer was met A. lumbricoides en/of T. trichiura. Skole in areas met die hoogste armoede syfer en tuberkulose annmeldings koers, het ook die hoogste prevalensie van helminte gehad. Alle kinders, ongeag hulle helmint status of hulle deelname in die studie, het gevolglik anti-helmintiese behandeling ontvang. BeG vaksinasie littekens en Mantoux vel toets response was beskikbaar op 'n subpopulasie van die bogenoemde skool kinders. Alhoewel dit aanvaar word dat die meerderheid van kinders BeG vaksinasie in die neonatale periode ontvang het, het slegs twee derdes van die kinders 'n BeG litteken getoon. Die resulatate dui daarop dat die prevalensie van BeG litteken positiwiteit, onafhanklik van ouderdom, laer was in kinders rondom die ouderdom van 11 jaar. In kontras met die konstante uitdrukking van BeG littekens, het die persentasie van Mantoux reaktiwiteit verhoog met ouderdom vanaf 13 % by 6 jaar tot 65 % teen 15 jarige ouderdom. Die tyd koers van Mantoux omskakeling met ouderdom dui daarop dat tuberkulin sensitiwiteit, geïnduseer deur BeG, afneem binne die eerste paar jaar van lewe en dat PPD responsiwiteit daarna deinduseer word deur omgewings blootstelling aan M. tuberculosis. In kontras met die ThllTh2 paradigma, was die prevalensie van helmint infeksies in kinders met 'n BeG litteken marginaal laer teenoor hulle sonder 'n litteken. 'n Relatiewe swak posititiewe korrelasie was gevind tussen tuberkulin responsiwiteit en helmint infeksie. Hierdie korrelasie was meer beduidend in kinders sonder 'n litteken. In hierdie sub-groep, was die helmintgeïnfekteerde kinders meer geneig om PPD responsief te wees teenoor hulle wat nie geïnfekteer was nie. Die data wys daarop dat omskakeling na PPD sensiwiteit kan lei tot infeksie met helminte. Die resultate stel voor dat die effek van helmint infeksie op die ontwikkeling van kliniese tuberkulose van so 'n aard is dat diegene met groot wurm ladings en wat goeie PPD response toon, meer geneig sal wees om weerstandig te wees. Diegene egter wat die parasiete beter kan beheer and wat goeie PPD response toon, sal meer geneig wees om vatbaar te wees vir tuberkulose. Die data dui ook daarop dat die BeG vaksien wat in die studie gebruik was, nie lei tot latente infeksie nie, terwyl patogene M tuberculosis dit wel doen en herhaardelik die immuun respons sal stimuleer. In 'n aparte studie, was dit gedemonstreer dat die gasheer-respons teen M. tuberculosis in pasiënte wat die gevaar loop om na suksesvolle voltooiing van behandeling, weer tuberkulose te ontwikkel, verskil van diegene wat beskermende immuniteit het. Onderhoude was ook gevoer met indiwidue wat deelgeneem het aan die studie, om ten einde hul sosiale en ekonomiese agtergrond te verstaan en hoe dit gekoppel is aan die siekte. Purified protein derivative (PPD) en M. tuberculosis-geinduseerde sitokien response was in die studie groepe bepaal. Die resultate wys daarop dat alhoewel 'n enkele immunologiese merker nie geidentifiseer kon word nie, was immunologiese patrone vir vatbaarheid welopgemerk. Indiwidue wat reeds een episode van tuberkulose gehad het en suksesvolle behandeling ontvang het, het 'n onderdrukte immuun profiel getoon. Dit het ingesluit hoë vlakke van die sitokiene, IL-lO en TGF-J3 asook hoë vlakke van serum IgE. Hierdie tipe profiel stel voor dat ten spyte van die vorige tuberkulose episode, hierdie persone nie beskermende immunitiet ontwikkel nie en dus vatbaar is vir herinfeksie en progressie tot siekte. Die onderhoude het getoon dat die meerderheid van mense in die studie populasie onder armoedige oorbevolkte omstandighede lewe, wat werkloosheid en ondervoeding insluit. Die studie het verder getoon dat hierdie indiwidue met die onderdrukte immuun profiel en wat in armoede lewe, in die nabye toekoms vatbaar is vir 'n tweede episode van tuberkulose. In die studie gemeenskap, met 'n tipiese derde wêreld opset, is daar gewys dat armoedeverwante faktore en helminte, mag bydra tot 'n dominante tipe 2 immuun respons wat op sy beurt, die beskermende tipe 1 response sal af-reguleer. Dit sal uiteindelik lei tot verhoogde vatbaarheid vir M. tuberculosis en uiteindelik progressie tot siekte (tuberkulose).

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Please see fulltext for abstract. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of the tuberculosis disease, is estimated to infect a third of the world’s population and is therefore, arguably, the most successful human pathogen in recorded history. Immense efforts to understand the genetic factors and biochemical processes underlying the complex interactions between M. tuberculosis and its host cells have delivered staggering insights into the profound proficiency by which this bacterium establishes and maintains an infection. It is now clear that M. tuberculosis can interfere with the immune responses initiated by host cells in such a manner as to subvert the various bactericidal conditions established by these cells and thus eliminate the tubercle bacilli that infect them. Specific characteristics of M. tuberculosis which provide it with this ability include a nearly impenetrable cell wall, secretion systems which secrete special factors which directly interact with host immune factors. This enables M. tuberculosis to modulate the activities of the host environment and unique metabolic adaptations of M. tuberculosis allows the organism to survive in the hypoxic, oxidative, nitrosative, acidic and nutrient poor environment of immune cell phagosomes and to persist for decades in a quiescent state in otherwise healthy people. New observations into the pathways which constitute energy, carbon and central nitrogen metabolism, among others, in M. tuberculosis, suggest that a carefully orchestrated homeostasis is maintained by the organism which may modulate the concentrations and ameliorate the effect of molecules that are important to defensive strategies employed by host cells. Here we discuss various recent studies as well as new information provided by this study, focusing on central metabolism and its regulation in M. tuberculosis. We aim to highlight the importance of nitrogen metabolism in the subversive response employed by M. tuberculosis to survive, colonise and persist in the host. We argue that the homeostatic regulation of nitrogen metabolism in M. tuberculosis presents a profound vulnerability in the pathogen which should be exploited with compounds that inhibit the activities of various effector proteins found in this pathway and that are unique to the organism. Such compounds may provide valuable novel chemotherapies to treat tuberculosis patients and may alleviate the burden of multiple drug resistance which plagues tuberculosis treatments. Specifically, in this study we investigate the role of M. bovis BCG glutamate dehydrogenase (GDH) and glutamate synthase (GltS) by subjecting knockout mutants of the aforementioned gene products to various cellular stress conditions. Furthermore, we investigated how the genomes of each M. bovis BCG strain was affected post deletion of the aforementioned protein products. The role of GDH was also tested in an murine macrophage model of infection to elucidate potential importance to colonisation and infection. This study provides novel results indicating an importance of GDH toward the resistance of nitrosative stress as well as a requirement for optimal persistence in RAW 264.7 macrophages. In addition, it was found that GltS is dispensable for resistance against nitrosative stress. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme wat die aansteeklike siekte tuberkulose veroorsaak, infekteer ongeveer ‘n dêrde van die wêreld populasie en is daarom, waarskynlik, een van die mees suksesvolle menslike patogene in geskiedenis. In die afgelope jare is daar noemenswaardige poging aangewend om genetiese faktore sowel as biochemiese prosesse te verstaan wat die komplekse interaksies tussen M. tuberculosis en sy gasheer selle verduidelik. Dit is nou voor die hand liggend dat M. tuberculosis kan inmeng met die reaksies van die immuun sisteem, om dus die bakteriosidiese omgewing wat geskep word deur die selle van hierde sisteem te vermy. Daar is spesifieke kenmerke van M. tuberculosis wat toelaat dat die bacilli so ‘n omgewing kan weerstaan. Hierdie kenmerke is, onder andere, ‘n byna ondeurdringbare selwand en uitskeiding sisteme wat spesiale faktore vrystel. Hierdie faktore het die vermoë om direk met die gasheer immuun sisteem ‘n interaksie te hê wat dus die immuun sisteem moduleer. Verder, is M. tuberculosis se metabolisme aan gepas om die organisme te help teen die lae suurstof, hoë oksidatiewe en stikstof stress, lae pH en lae voedingswaarde omgewing te oorleef. M. tuberculosis het ook die vermoë om vir ‘n onbeperkte tyd in ‘n statiese toestand te oorleef, in gashere wat toon as gesond. Nuwe waarnemings in die energie, koolstof en sentrale stikstof metaboliese paaie stel voor dat ‘n homeostase gehandhaaf word deur M. tuberculosis, wat die konsentrasies van verskeie molekules moduleer of die effek van molekules wat deur die gasheer vrygestel word as ‘n verdedigings meganisme versag. In hierdie dokument bespreek ons verskeie studies, asook nuwe inligting voortgebring deur hierdie studie, wat fokus op sentrale metabolisme en sy regulering in M .tuberculosis. Ons raak aan die vermoë van M. tuberculosis om intrasellulêr te oorleef, koloniseer en voort te bestaan in ‘n gasheer. Ons vemoed dat die homeostatiese regulering van stikstof metabolisme in M. tuberculosis n diepgaande kwesbaarheid in die patogeen skep wat die potentiaal het om uit gebuit te word. Molekules kan gesintiseer word wat die aktiwiteite van verskeie ensieme in hierdie padweg inhibeer en sodoende die organisme hinder. Sulke molekules mag dalk as waardevolle en oorspronklike medisynes ontwikkel word om tuberkulose patiënte meer suksesvol te behandel asook om die las van middelweerstandige bakterieë te verlig. Met betrokke tot hierdie spesifieke studie, het ons die rol van glutamaat dehidrogenase (GDH) en glutamaat sintase (GltS) van M. bovis BCG bestudeer deur om die uitslaan mutante van die genoemde geen produkte aan verskeie sellulêre stress toestande bloot te stel. Die effek van die verlore gdh en gltBD gene op die evolusie van die genome van elke M. bovis BCG uitslaan mutant ras ten opsigte van die wilde tipe was ook bestudeer. Die rol van GDH was getoets in ‘n muis makrofaag model van infeksie om te bepaal of GDH n funksie het in koloniseering en infeksie van M. bovis BCG. Hierdie studie het nuwe bevindinge voort gebring wat die belangrikheid van GDH in die weerstand teen stikstof oksied stress. Daar is verder bevind dat GDH n vereiste toon vir die suksessvolle oorlewing van M. bovis BCG in RAW 264.7 macrofage

Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Drug-resistant tuberculosis poses a threat to global tuberculosis control by the WHO DOTS strategy. Studies in the United States and Europe have shown (i) that drug-resistant tuberculosis is present in every country; (ii) that, by contrast to previous dogma, drug-resistant bacilli are virulent and can be transmitted, especially in institutional settings and to immunocompromised patients; and (iii) that the majority of cases arise by acquisition of drug resistance due to errors in the management of TB cases. (iv) Furthermore, it has been shown that the extremely high case fatality rates of the 1980s and early 1990s can be reduced by individualized, but costly treatment. However, the majority of drug-resistant TB cases reside in the developing world. Data on disease epidemics in less developed parts of the world are scarce. The aim of this thesis was to study the disease dynamics of drug-resistant TB in a developing country where TB is endemic. All cases of drug-resistant TB during a 5-year period in two communities with poor socioeconomic living conditions were included for this observational study. Three different methods were used: restriction fragment length polymorphism (RFLP), mutation detection analysis by dot-blot hybridisation technique and a Geographic Information System. Results of RFLP analysis and mutation detection analysis showed that community outbreaks of drug-resistant Mycobacterium tuberculosis strains occur, even without the involvement of immunocomprimised patients. Infection with a drug-resistant strain occurred in new patients (primary drug resistance) as well as in patients treated before (exogenous reinfection). Exogenous reinfection was also shown to be an important mechanism of recurrence after previous cure for drug-sensitive TB. Transmission of drug-resistant strains occurred more frequent in areas with lower socioeconomic living conditions. The relative contribution of transmission differed substantially between the group of multi drugresistant (two thirds of cases) and single-drug-resistant (no cases) cases, which probably reflects the prolonged infectiousness of multi drug-resistant cases. To stop the growing epidemic of multi drug-resistant TB, prevention of acquisition as well as transmission of drug-resistant tuberculosis will be required. This will only be possible in areas where a DOTS strategy is well functioning and with a modification of central elements of the standard DOTS mechanism: a "DOTS-plus" strategy. Early and accurate diagnosis of drug resistance is essential for effective management. Diagnosis based on two direct smear tests might have to be replaced by routine drugsusceptibility tests at diagnosis. Because the routine performance of phenotypic drugsusceptibility tests was inferior to the performance of genotypic tests, the development of an affordable commercial kit testing a limited number of mutations conferring resistance could be of great value in the global fight against multidrugresistant TB. Because of the importance of early diagnosis, selective active contact tracing for multidrug-resistant cases, additional to the routine passive contact tracing, could prove to be cost-effective. Individualized treatment regimens are effective in reducing the failure rate, mortality and probably transmission of multidrug-resistant TB. Multidrug-resistant tuberculosis is a problem confronting the efforts for global tuberculosis control. Efficient strategies to turn the tide exist, but international political commitment and financial support will be essential. / AFRIKAANSE OPSOMMING: Middel weerstandige tuberkulose hou 'n bedreiging in vir globale tuberkulose kontrole deur die WGO DOTS strategie. Studies in die Verenigde State en Europa het getoon (i) dat middel weerstandige tuberkulose in alle lande voorkom; (ii) dat, in teenstelling met vorige dogma, middel weerstandige bakterieë virulent is en oorgedra kan word, veral in inrigtings en aan immuun-onderdrukte pasiënte; en (iii) dat die meeste gevalle ontstaan deur die verwerwing van middel weerstandigheid a.g.v. die foutiewe hantering van tuberkulose gevalle. (iv) Bykomend is getoon dat die ontsettende hoë mortaliteit syfers van die 1980s verlaag kan word deur geindividualiseerde, maar duur behandeling. Die meeste middel weerstandige tuberkulose gevalle woon egter in die ontwikkelende wêreld. Data oor siekte epidemies in minder ontwikkelde dele van die wêreld is skaars. Die doel van hierdie tesis was om die siekte dinamiek van middel weerstandige tuberkulose te bestudeer in 'n ontwikkelende land waar tuberkulose endemies is. Alle gevalle van middel weerstandige tuberkulose gedurende 'n 5-jaar periode in twee lae sosio-ekonomiese gemeenskappe, is in hierdie studie ingesluit. Drie verskillende metodes is gebruik: restriksie fragment lengte polimorfisme (RFLP), mutasie analise deur dot-blot hibridisasie en 'n Geografiese Inligting Stelsel. Resultate van die RFLP analise het getoon dat uitbrake van middel weerstandige Mycobacterium tuberculosis stamme in die gemeenskap voorkom, selfs sonder die aantasting van immuun-onderdrukte pasiënte. Infeksie met middel weerstandige stamme het voorgekom in nuwe pasiënte (primêre middel weerstandigheid) en ook in pasiënte wat reeds voorheen behandel is (eksogene herinfeksie ). Daar is ook gevind dat eksogene herinfeksie 'n belangrike meganisme was van herhaalde tuberkulose na vorige genesing van middel sensitiewe tuberkulose. Die oordrag van middel weerstandige stamme het meer dikwels voorgekom in areas met laer sosioekonomiese omstandighede. Die relatiewe bydrae van oordrag het merkwaardig verskil tussen multi-middel weerstandigheid (twee derdes van gevalle) en enkelmiddel weerstandigheid (geen gevalle). Dit weerspieël waarskynlik die verlengde periode van infektiwiteit van die multi-middel weerstandige gevalle. Die bekamping van die groeiende epidemie van multi-middel weerstandige tuberkulose, vereis die voorkoming van verworwe sowel as oorgedraagde middel weerstandige tuberkulose. Dit sal slegs moontlik wees in areas waar 'n DOTS strategie reeds goed funksioneer en met 'n aanpassing van die sentrale elemente van die roetine DOTS meganisme: 'n "DOTS-plus" strategie. Vroeë en akkurate diagnose van middel weerstandigheid is essensieël vir effektiewe hantering. Diagnose gebaseer op twee direkte sputum smeer toetse mag moontlik vervang moet word deur roetine middel sensitiwiteit bepalings by diagnose. Die roetine fenotipiese middel sensitiwiteit bepaling is gevind om minderwaardig te wees in vergelyking met die genotipiese toetse. Die ontwikkeling van 'n bekostigbare toetsstelsel wat die mees algemene mutasies vir middel weerstandigheid sal opspoor, kan van groot waarde wees in die stryd teen mutimiddel weerstandige tuberkulose. Aangesien vroeë diagnose so belangrik is, kan aktiewe kontak opsporing koste-effektief wees. Ge-individualiseerde behandelingskedules is effektief om die sukses van behandeling en oorlewing te verbeter, en moontlik ook om die oordrag van multi-middel weerstandige tuberkulose te verminder. Multi-middel weerstandige tuberkulose is 'n probleem vir die globale kontrole van tuberkulose. Effektiewe strategieë om die vloed te stuit, bestaan, maar politieke verbintenis en geldelike ondersteuning sal essensieël wees.

Thesis (MSc MedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Background The QuantiFERON In-Tube (QFT IT) assay is an Interferon-gamma release assay (IGRA) which is currently used to detect Mycobacterium tuberculosis (M. tb) infection. It however cannot differentiate between latent infection and active tuberculosis (TB) disease. In an attempt to improve this tool to accurately diagnose active TB, the release of a variety of markers should be assessed in combination with Interferon gamma (IFN- γ). Luminex analysis was previously done on QFT plasma and promising candidates were identified which could be of great value in treatment response studies. IFN-γ ELISpot, are not only used to detect M.tb infection, but is also implicated in vaccine trails to assess immunogenicity. The IFN-γ ELISpot and flow cytometry are the most common assays to assess these phenomena during clinical trials. Our aim therefore was to develop a multi platform immune analysis assay using the QFT IT system. Study design and method The first approach of this study was to optimize the QFT IT assay for flow cytometry applications. The following questions formed part of the optimization study: How does the QFT whole blood assay (QFT-WBA) compare to the currently used WBA? Is antigen re-stimulation required after the initial incubation time and for how long should cells be re-stimulated in the presence of Brefeldin A? The second approach was to use the optimized QFT-WBA for community controls (CTRL), household contacts (HHC) and TB cases, which were recruited from the high TB incidence areas Ravensmead, Uitsig and Elsies River. The infection status of each participant was determined by IFN-γ ELISA and Luminex analysis was performed to measured wide range of cytokine expression. In addition immune cell markers like CD14, CD4, CD8, CD19, and T cell receptor gamma delta (TCRγδ) were characterized; polyfunctional characteristics (IFN-γ, Tumor necrosis factor-alpha (TNF-α) and Interleukin-2 (IL-2)) and proliferation (Ki-67+) of T cells determined by flow cytometry. Results After stimulating the whole blood of the study participants for 22 hours with the M. tb specific antigens, early secreted antigenic target 6 kDa (ESAT-6), culture filtrate protein-10 kDa (CFP-10) and TB7.7 the levels of TNF-α producing CD4 T cells were elevated in TB cases compared to HHCs. After stimulating the whole blood for 6 days TNF-α producing T cells declined in TB cases and HHC showed a higher expression. CD40L+CD4+ (p=0.0225) was increased in HHC while IL-9+CD8+ (0.3230) was decreased in HHC compared to TB cases. Other markers such as IL-5(AG-NIL), IL-13(Ag- NIL), FGF basicAg, GM-CSFNIL, VEGFNIL/(Ag-NIL), MIP-1βAg and MCP-1Ag/(Ag-NIL) showed significant differences between HHC and TB cases. Conclusions The responses in the QFT-based assay were generally comparable to the WBA that is routinely used. The differences of TNF-α expression seen in QFT-WBA and QFTLPA could be explained by the fact that effector T cell responses were measured in the short term assay and the central memory T cell responses in the long term assay. Our study therefore shows that the QFT-based tests can be used to simultaneously assess a wide range of immunological markers and not only IFN-γ expression. / AFRIKAANSE OPSOMMING: Agtergrond Die QuantiFERON In Tube (QFT IT) toets is ‘n Interferon-gamma vrystellingstoets (IGRA) wat huidiglik dien as ‘n maatstaf van Mycobacterium tuberculosis (M. tb) infeksie. Hierdie toets kan egter nie onderskei tussen latente infeksie en aktiewe tuberkulose (TB) nie. ‘n Noemenswaardige verbetering in die vermoë van hierdie toets om aktiewe TB te diagnoseer, berus op die studie van ‘n verskeidenheid vrygestelde merkers, insluitend Interferon gamma (IFN-γ). In vorige Luminex studies op QFT plasma, is belowende kandidate geïdentifiseer wat van groot waarde kan wees vir studies wat fokus op die reaksie tot behandeling. Die IFN-γ ELISpot dien nie net as ‘n maatstaf van M.tb infeksie nie, maar word ook in vaksienproewe betrek om die aard van immuniteit te ondersoek. Die IFN-γ ELISpot toets sowel as vloeisitometriese toetse, is van die mees algemene toetse om hierdie verskynsels te meet, tydens kliniese proewe. Die doel van hierdie studie was dus om die QFT IT sisteem te ontwikkel as ‘n basis vir ‘n multiplatform immunologiese analiseringstoets. Studie ontwerp en metode Die inleidende benadering van hierdie studie was die optimisering van die QFT IT toets, vir vloeisitometrie doeleindes. Die volgende vrae het deel uitgemaak van die optimiseringstudie: Hoe vergelyk die QFT heelbloedtoets (QFT-WBA) met huidige WBAs wat in gebruik is? Word meermalige antigeenstimulasies benodig na die oorspronklike inkubasieperiode en hoe lank moet die tydperk wees vir sellulêre opvolgstimulasie, in die teenwoordigheid van Brefeldin A? As ‘n tweede benadering, was om die geoptimiseerde QFT-WBA te gebruik vir gemeenskapskontroles (CTRL), huishoudelike kontakte (HHC) en TB gevalle. Al drie hierdie groepe was opgeneem uit Ravensmead, Uitsig en Elsies Rivier, areas met betreklik hoë vlakke van TB infeksie. Elke persoon in die studie se vlak van infeksie is vasgestel met behulp van die IFN-γ ELISA en Luminex analiese was uitgevoer, om ‘n wye verskeidenheid uitdrukkingsvlakke van sitokiene te meet. Dies meer, was immuunselmerkers soos CD14, CD4, CD8, CD19 en T sel reseptor gamma delta (TCRγδ) gekarakteriseer. Meervuldige funskionele karakteristieke (IFN-γ, Tumor nekrose faktor-alpha (TNF-α) en Interleukin-2 (IL-2)) en vermenigvuldiging van T-selle, was vasgestel deur middel van vloeisitometrie. Resultate Nadat die heelbloed van studiedeelnemers gestimuleers was met M. tb spesifieke antigene, vroeë afskeidings antigeniese teiken 6kDa (ESAT-6), kultuurfiltraatproteïn 10kDa (CFP-10) en TB7.7, vir 22 uur, was gevind dat vlakke van TNF-α produserende CD4 T selle hoër was in TB pasïente, in vergelyking met HHCs. Nadat die heelbloed vir 6 dae gestimuleer was, het die vlak van TNF-α produserende T-selle afgeneem in TB pasïente, terwyl dit hoër was in HCC. CD40L+CD4+ (p=0.0225) het hoër vlakke bereik in HHC, terwyl IL-9+CD8+ (0.3230) vlakke afgeneem het, in vergelyking met TB pasïente. Ander merkers soos,onder andere, IL-5(AG-NIL), IL-13(Ag-NIL), FGF basicAg, GMCSFNIL, VEGFNIL/(Ag-NIL), MIP-1βAg and MCP-1Ag/(Ag-NIL), het noemenswaardige verskille geopenbaar tussen HHC en TB pasïente.

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.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy