Development of immune-based TB tests suitable for resource limited settings

Thesis (PhD)--Stellenbosch University, 2014.ENGLISH ABSTRACT: - Background - Tuberculosis (TB) is still one of the leading causes of death in poor socio-economic settings. This situation is encouraged by the lack of simple and rapid tests suitable for rapid diagnosis. The newly developed Interferon-gamma Release assays (IGRAs) can detect Mycobacterium tuberculosis (M.tb) infection but fail to discriminate active TB from latently infected individuals. - Objectives - The present thesis aims to develop a rapid and simple test for the diagnosis of active TB disease. This objective was divided into four sub-objectives: 1) identification of potential M.tb antigens and host markers suitable for a TB test using a 7-day whole blood assay (WBA), 2) validate the promising results in an overnight WBA for a rapid, albeit not ex vivo, test, 3) evaluate the diagnostic utility of a two colour ELISpot test, 4) use an unbiased approach to discover multiple new host markers with diagnostic utility using mass spectrometry. - Methods and results - Participants were recruited from the Ravensmead/Uitsig community and day clinics. Stimulated and unstimulated analyte levels in 7-day and overnight WBA supernatants from active TB cases were compared to analyte levels in controls. The results of these experiments showed that Rv0081-stimulated levels of IP-10, IL-12p40, TNF-α and IL-10 were the most promising diagnostic markers in the long term assay as they could correctly classify 100% of the study participants in this assay. Acute phase proteins mainly CRP and SAA were the best diagnostic antigens in the short term assay. The diagnostic utility of these markers was greater in Quantiferon Nil supernatants compared to the stimulated samples. IFN-γ and IL-2 ELISpot was performed where it was found that single cytokine measures could not discriminate active TB to latent infection. When single and double secreting cell populations were taken into consideration, a combination model of ESAT6/CFP10-stimulated single IFN-γ, single IL-2 and IFN-γ/IL-2 double secreting cells could classify participants into their clinical groups with good accuracy. In a pilot study for future discovery of diagnostic markers by mass spectrometry, three depletion methods (ProteoSpin column, Heparin column and ProteoPrep 20) were assessed to identify the most appropriate depletion method for high abundant proteins from serum. The depleted serum samples were analysed in Orbitrap Velos. The antibody based method, ProteoPrep 20, was the best depletion method as it led to the visualisation of a larger number of proteins on Orbitrap. - Conclusion - M.tb antigen-stimulated host markers hold promises in diagnosis of active TB disease. The excellent accuracy observed in the long term assay could not be repeated in the short term assay. Acute phase proteins are the most promising but perform better in unstimulated than in stimulated supernatants and should be evaluated in ex vivo samples like serum or plasma. However, it is likely that further unbiased proteomic approaches, like mass spectrometry, will identify additional promising markers that will allow the development of ex vivo, accurate, pointof- care tests for TB.AFRIKAANSE OPSOMMING: - Agtergrond - Tuberkulose (TB) is steeds die hoof oorsaak van meeste sterftes in behoeftige gebiede wêreldwyd. Hierdie situasie word aangemoedig deur die gebrek aan ʼn eenvoudige en vinnige diagnostiese toets wat spesifiek toepaslik is vir hierdie gebiede. Die nuut ontwikkelde ʽInterferon Gamma-Release’ (IGRA) toetse kan met uitstekende akkuraatheid ʼn Mycobacterium tuberkulose (M.tb) infeksie opspoor, maar is ondoeltreffend om tussen aktiewe TB en sluimerende M.tb infeksie in die mens te onderskei. - Objektiewe - Die huidige tesis het ten doel om 'n vinnige en eenvoudige toets vir die diagnose van aktiewe TB te ontwikkel. Hierdie doelwit is in vier sub-doelwitte verdeel: 1) identifikasie van potensiële M.tb antigene en gasheer merkers wat geskik vir 'n TB-toets deur gebruik te maak van ʼn 7-dag vol bloed toets (WBA), 2) evaluasie van die toepaslikheid van hierdie potensiële merkers en antigene in ʼn oornag WBA, vir die ontwerp van in 'n direkte toets, 3) beoordeling van die diagnostiese waarde van die twee-kleur ELISpot, 4) Assessering van die diagnostiese doeltreffendheid van verskeie gasheer merkers deur gebruik te maak van massaspektrometrie. - Metodes en Resultate - Deelnemers is gewerf vanuit die Ravensmead / Uitsig gemeenskap en klinieke. Gestimuleerde en ongestimuleerde analiet vlakke in 7-dag- en oornag WBA supernatante van aktiewe TB-pasiënte is vergelyk met analiet vlakke in ooreenstemmende kontrole groepe. Resultate van hierdie eksperiment het getoon dat vlakke van IP-10, IL-12p40, TNF-α en IL-10 in antigeen Rv0081 gestimuleerde supernatante, die mees belowende diagnostiese merkers in die lang termyn toets is. Hierdie merkers kon met 100% akkuraatheid die studie deelnemers klassifiseer. Akute fase proteïene, hoofsaaklik CRP en SAA, is aangewys as die beste diagnostiese merkers in die kort termyn toets. Die diagnostiese waarde van hierdie merkers was meer omvangryk in Quantiferon Nil supernatante in vergelyking met dié van WBAs. IFN-γ/IL-2 twee-kleur ELISpot is uitgevoer volgens die vervaardiger se instruksies. Direkte vergelyking het aangetoon dat die kol-vormende eenhede vanaf individuele sitokien produserende selle, nie kan diskrimineer tussen aktiewe TB te latente M.tb infeksie nie. Alhoewel, indien beide enkel-en dubbel sitokien produserende sel populasies in ag geneem word, kan 'n kombinasie van die model ESAT6/CFP10-stimuleerde enkel IFN-γ, enkel IL-2 en IFN- γ/IL-2 dubbel produserende selle, deelnemers klassifiseer in hul kliniese groepe met goeie akkuraatheid. Drie metodes (ProteoSpin kolom Heparien kolom en ProteoPrep 20) is gebruik om oorvloedige serum proteïene te vernietig, waarna die diagnostiese nut van sirkulerende serum merkers deur middel van massaspektrometrie bepaal is. Analise van die serum monsters met behulp van die Orbitrap Velos, het aangetoon dat die teenliggaam metode, ProteoPrep 20, die mees suksesvolle metode is, aangesien dit gelei tot die visualisering van 'n groter aantal proteïene op die Orbitrap. - Gevolgtrekking - M.tb antigeen gestimuleerde gasheer merkers toon groot potensiaal in die diagnose aktiewe TB. Die uitstekende diagnostiese akkuraatheid wat waargeneem is in die lang termyn toets kon egter nie met dieselfde graad van akkuraatheid in die kort termyn toets herhaal word nie. Akute fase proteïene is bewys as die mees belowende ongestimuleerde merkers in die kort termyn toets. Daarbenewens verhoog die diagnostiese waarde van akute fase proteïne aansienlik wanneer gemeet word in Quantiferon supernatant in vergelyking met vol bloed supernatant