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

[摘要] 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 Mycobacteriumtuberculosis (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 antigensand host markers suitable for a TB test using a 7-day whole blood assay (WBA), 2) validate thepromising results in an overnight WBA for a rapid, albeit not ex vivo, test, 3) evaluate thediagnostic utility of a two colour ELISpot test, 4) use an unbiased approach to discover multiplenew host markers with diagnostic utility using mass spectrometry. - Methods and results - Participants were recruited from the Ravensmead/Uitsig community and day clinics. Stimulatedand unstimulated analyte levels in 7-day and overnight WBA supernatants from active TB caseswere compared to analyte levels in controls. The results of these experiments showed thatRv0081-stimulated levels of IP-10, IL-12p40, TNF-α and IL-10 were the most promisingdiagnostic markers in the long term assay as they could correctly classify 100% of the studyparticipants in this assay. Acute phase proteins mainly CRP and SAA were the best diagnosticantigens in the short term assay. The diagnostic utility of these markers was greater inQuantiferon Nil supernatants compared to the stimulated samples.IFN-γ and IL-2 ELISpot was performed where it was found that single cytokine measures couldnot discriminate active TB to latent infection. When single and double secreting cell populationswere 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 clinicalgroups with good accuracy. In a pilot study for future discovery of diagnostic markers by mass spectrometry, three depletionmethods (ProteoSpin column, Heparin column and ProteoPrep 20) were assessed to identify themost appropriate depletion method for high abundant proteins from serum. The depleted serumsamples were analysed in Orbitrap Velos. The antibody based method, ProteoPrep 20, was thebest 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. Theexcellent 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 instimulated 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, willidentify additional promising markers that will allow the development of ex vivo, accurate, pointof-care tests for TB.

[发布机构] Stellenbosch University