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.