Immunological markers for active TB and early treatment response indicators

Awoniyi, Dolapo Olaitan (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

Background The difficulty in diagnosing tuberculosis (TB) and evaluating TB treatment response are two major problems that are hampering the defeat of this infectious disease. The current TB diagnostic tools have several limitations and these call for the development of a simple, rapid and accurate diagnostic test that is suitable for use in poor-resource settings. Aim and Objectives This thesis aims to identify host markers for the development of a rapid and simple test for TB diagnosis and for monitoring early TB treatment response. The objectives are: 1. To investigate the diagnostic accuracy of host markers detected in Mycobacterium tuberculosis (Mtb) antigen-stimulated overnight whole blood culture supernatant. 2. To investigate the profiles of inflammatory markers of active TB patients undergoing treatment in a 14-day EBA trial for treatment monitoring potential. 3. To investigate the combined performance of the responses of IgG, IgM and IgA to selected mycobacterial antigens for their diagnostic potential. Methodology Participants were recruited as part of the recently concluded EDCTP-funded AE-TBC study and a 14-day phase II randomised clinical trial (early bactericidal activity (EBA) study of seven treatment arms). Sputum and blood samples were collected at different time points and multiplex cytokine array analysis performed on plasma or serum samples by Luminex and anti-mycobacterial antibodies detected by ELISA. Results After overnight stimulation of whole blood with ESAT-6/CFP-10, RV0081, Rv1284 and Rv2034, the most promising diagnostic markers were CRP, Ferritin, SAA and IP-10. Unstimulated host markers yielded the best discriminatory power. A six-marker biosignature comprised mostly of unstimulated cytokine levels shows promise for active TB diagnosis. Stellenbosch University https://scholar.sun.ac.za iii There were significant changes for CRP, IL-6, VEGF, sIL-2Rα, Ferritin, and sTNFRII from baseline to end of 14 day EBA evaluation in several treatment arms. However, none of these markers mirrored the decrease in the measured bacterial load in sputum. A four-marker combination only accounted for 20% of the variation in observed in both TTP and CFU. The highest sensitivity and specificity was obtained with anti-16 kDa IgA (95%/95%) and anti-MPT64 IgA (95%/90%). A higher accuracy was obtained with a 3 or 4 antibody combination. Anti-16 kDa IgA and anti-16 kDa IgM, decreased significantly while anti-LAM IgG and anti-TB-LTBI IgG increased significantly at the end of month six anti-TB treatment. Conclusion Host biomarkers hold promise as a diagnostic tool in TB disease. In spite of the moderate accuracy of Mtb antigen-stimulated host markers, these could still have value in difficult to diagnose TB, like paediatric TB or extrapulmonary TB, and should be evaluated in future studies. Although host markers only explained a small degree of the variation in bacterial measures in early bactericidal activity studies, their potential role in overall treatment effect remains to be investigated. Serodiagnostic markers against novel Mtb antigens showed potential for future development into a simpler format for use at the point-of-care. These results should be validated in large scale studies in appropriate participant groups.

Agtergrond Probleme met TB diagnosering en evaluering van die reaksie op TB behandeling is twee groot struikelblokke wat die effektiewe behandeling strem. Die huidige TB diagnostiese toetse het verskeie tekortkominge en dit is belangrik om ‘n eenvoudige, vinnige en akkurate diagnostiese toetse te ontwikkel wat effektief in hulpbron-beperkte gemeenskappe gebruik kan word. Doelwitte Die doel van die studie was om gasheer biomerkers te identifiseer vir die ontwikkeling van ‘n vinnige en eenvoudige TB diagnostiese toets en die waarneming van die reaksie op vroeë TB behandeling. Die doelwitte is: 1. Om die diagnostiese akkuraatheid van die gasheer biomerkers in ‘n Mycobacterium tuberculosis (MTB) antigeen-gestimuleerde bloed kultuur te bepaal. 2. Om die profiel van inflammatoriese biomerkers van pasiënte met aktiewe TB in ‘n EBA proef te analiseer. 3. Om die gebruik van IgG, IgM en IgA op geselekteerde mycobacterial antigene vir diagnostiese potensiaal te bepaal. Metodes Deelnemers was gewerf en deel van die onlangse gefinaliseerde EDCTP-befondsde, AE- TBC studie en die 14-dae fase-twee kliniese ewekansige proef (EBA-studie met sewe behandelings arms). Sputum en bloed monsters is op verskillende tye versamel, multiplex sitokinien, verskeidenheid ontleding is deur Luminex op plasma en serum monsters gedoen en anti-mycobacterial teenliggame is deur ELISA geïdentifiseer. Resultate Nadat die bloed oornag met ESAT-6/CFP-10, RV0081, Rv1284 en Rv2034 gestimuleer was, het CRP, Ferritin, SAA and IP-10 as die mees belowende diagnostiese merkers na vore gekom. Ongestimuleerde gasheer merkers het die beste onderskeidings vermoë getoon. Die ses-merker bio-kenmerk bestaan hoofsaaklik uit ongestimuleerde sitokiene wat se vlakke die belowendste resultate vir die diagnosering van aktiewe TB opgelewer het. Noemenswaardige veranderinge was vir CRP, IL-6, VEGF, sIL-2Rα, Ferritin en sTNFRII gesien teen oor die basislyn tot en met die einde van die 14 dag EBA evaluasie in verskeie van die behandelings afdelings. Alhoewel geeneen van die merkers die afname in die bakteriële lading in sputum weerspieël het nie. ’n Vier-merker kombinasie kon slegs vir 20% van die variasie in beide TTP en CFU verantwoord. Die beste sensitiwiteit en spesifisiteit was behaal deur die anti-16 kDa IgA (95%/95%) en anti-MPT64 IgA (95%/90%). ’n Hoër akkuraatheid is met ’n kombinasie van 3 of 4 teenliggame verkry. Anti-16 kDa IgA en anti- MPT64 IgA vlakke het beduidend afgeneem terwyl anti-LAM IgG en anti-LTBI IgG weer beduidend toe geneem het teen die einde van ’n ses maande anti-TB behandeling. Gevolgtrekkings Gasheer biomerkers as diagnostiese instrumente vir die toets van TB lyk belowend. Ten spite van die middelmatige akkuraatheid van Mtb antigeen-stimuleerde gasheermerkers mag hulle wel waarde hê in moeilik diagnoseerbare TB, soos kindertuberkulose of ekstapulmonale tuberkulose, en behoort verder evalueer te word in toekomstige studies. Alhoewel die gasheer merkers net vir ’n klein persentasie van die variasie in bakteriële lading kon verklaar in vroeë bakteriële dodingstudies, is daar ’n potensiële rol in die meet van algemene behandelings uitkomste wat verdere ondersoek benodig. Serologiese diagnostiese merkers teen nuwe Mtb antigene toon om belowend te wees vir die ontwikkeling van ’n eenvoudige punt van sorg toestel. Die resultate moet geëvalueer word in groter skaal studies in toepaslike deelnemergroepe.

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