Analysis of ex vivo host biomarkers in sputum samples for diagnosis of pulmonary tuberculosis

Mendy, Joseph F. (2019-12)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

Background Despite all the interventions deployed to control tuberculosis (TB), the disease still continues to be the principal cause of death from a single infectious agent in resource constrained settings. An estimated 60% of suspected TB patients do not have access to TB diagnostic tests. With the limitations of the current diagnostic tests and the importance of early diagnosis and initiation of treatment, biomarker diagnosis of TB would be an optimal option. For biomarkers are indicators of immune activity and state. Therefore, host or pathogen biomarker of TB disease would be ideal. Hence, the aim of this project was to profile a broad array of host markers for development of optimal signatures for detection of pulmonary tuberculosis from other respiratory disorders using ex vivo sputum samples Methods We recruited patients who were seeking medical attention at the MRCG at LSHTM outpatients department and Tuberculosis clinic with symptoms suggestive of TB, prior to clinical or microbiological diagnosis. All age groups were recruited. Sputa were collected at baseline from all participants and at 1 and 2 months from the confirmed TB cases. The sputa were digested with Sputolysin and the supernatant analysed using Luminex arrays while RNA extracted from the pellet were analysed with RT-qPCR. Statistical analyses and graphs were generated using R programming Language and GraphPad Prism, with a q value ≤ 0.05 considered significant. A receiver operating curve (ROC) was used to assess the diagnostic performance of individual and combination markers. Results Confirmed TB (428) and ORD (313) patients were analysed, 70 markers were assessed for diagnostic potential and treatment response. Of these, 37 were significantly different between TB and ORD. The best single marker was MMP-2 with an AUC of 0.73. An eight-marker signature (IFNү, IL-1β, IL-8, IL-10, IL-12p70, MIP-1β, RANTES and VEGF) was able to diagnose smear and culture positive TB from ORD with an AUC of 0.77, sensitivity of 78% and specificity of 70%, while a threemarker signature (IL-1β, IL-7 and VEGF) classified smear negative but culture positive TB from ORD with an AUC of 0.74, sensitivity of 86% and specificity of 60%. Among children who had TB, a fourmarker signature (FGF, IL-4, MIP-1a and RANTES) differentiated those with TB from ORD, with an AUC of 0.87, sensitivity of 82% and specificity of 87% and a five-marker signature consisting of BAFF, C3L1, IL-22, MMP-3 and sTNFR1 was able to discriminate TB and HIV co-infected from ORD with an AUC of 0.90, sensitivity of 88% and specificity of 85%. We also found a four-marker signature consisting of EGF, IL-15, MIP-1β and TNF-β that could predict slow versus fast treatment responders at baseline with an AUC of 0.74, sensitivity of 75% and specificity of 80%. Conclusion We have discovered novel sputum host biomarkers and biosignatures for screening of tuberculosis and treatment response. The data is promising for potential translation into a user friendly device as a rapid screening test for pulmonary TB. However, this markers and signatures require further investigations to authenticate their usefulness.

Agtergrond Ten spyte van al die ingrypings wat toegepas word om tuberkulose (TB) te beheer, bly die siekte steeds die grootste oorsaak van die dood van 'n enkele aansteeklike middel in beperkte hulpbronne. Na raming het 60% van die vermoedelike TB-pasiënte nie toegang tot diagnostiese toetse vir TB nie. Met die beperkinge van die huidige diagnostiese toetse en die belangrikheid van vroeë diagnose en inisiëring van behandeling, sou die biomerk-diagnose van TB 'n optimale opsie wees. Vir biomerkers is dit 'n aanduiding van immuunaktiwiteit en toestand. Daarom is gasheer- of patogeenbiomerker van TB-siekte ideaal. Die doel van hierdie projek was dus om 'n wye verskeidenheid gasheermerkers te profiel vir optimale handtekeninge vir die opsporing van pulmonale tuberkulose van ander respiratoriese afwykings met behulp van ex vivo sputum monsters. Metodes Ons het pasiënte gewerf wat mediese behandeling by die MRCG by LSHTM-buitepasiënte-afdeling en tuberkulosekliniek opgedoen het met simptome wat dui op TB, voor die kliniese of mikrobiologiese diagnose. Alle ouderdomsgroepe is gewerf. Sputa is vanaf die beginlyn by alle deelnemers versamel en op 1 en 2 maande van die bevestigde TB-gevalle. Die sputa is met Sputolysin verteer en die supernatant is met behulp van Luminex-skikkings geanaliseer terwyl RNA wat uit die korrel onttrek is, met RT-qPCR geanaliseer is. Statistiese ontledings en grafieke is gegenereer met behulp van Rprogrammeringstaal en GraphPad-prisma, met 'n q-waarde ≤ 0,05 wat as beduidend beskou is. 'N Ontvangerbedryfskurwe (ROC) is gebruik om die diagnostiese prestasie van individuele en kombinasiemerkers te beoordeel. Resultate Bevestigde TB (428) en ORD (313) pasiënte is geanaliseer, 70 merkers is beoordeel vir diagnostiese potensiaal en reaksie op die behandeling. Hiervan was 37 aansienlik verskillend tussen TB en ORD. Die beste enkele merker was MMP-2 met 'n AUC van 0,73. 'N Handtekening met 'n agt merker (IFNү, IL-1β, IL-8, IL-10, IL-12p70, MIP-1β, RANTES en VEGF) was in staat om die smeer- en kultuurpositiewe TB van ORD te diagnoseer met 'n AUC van 0.77 , sensitiwiteit van 78% en spesifisiteit van 70%, terwyl 'n drie-merkteken (IL-1β, IL-7 en VEGF) die negatiewe maar kultuurpositiewe TB van ORD met 'n AUC van 0.74, 'n sensitiwiteit van 86% en die spesifisiteit van 60%. Onder kinders wat TB gehad het, het 'n vierteken-handtekening (FGF, IL-4, MIP-1a en RANTES) diegene met TB van ORD onderskei, met 'n AUC van 0,87, 'n sensitiwiteit van 82% en die spesifisiteit van 87% en 'n vyf- merkerhandtekening bestaande uit BAFF, C3L1, IL-22, MMP-3 en sTNFR1 was in staat om TB en MIV wat mede-besmet was van ORD te onderskei met 'n AUC van 0,90, 'n sensitiwiteit van 88% en die spesifisiteit van 85%. Ons het ook 'n vierteken-handtekening gevind wat bestaan uit EGF, IL-15, MIP-1β en TNF-β wat reageer op 'n trae versus vinnige behandeling by die basislyn met 'n AUC van 0,74, 'n sensitiwiteit van 75% en die spesifisiteit van 80%. Afsluiting Ons het nuwe sputum gasheer-biomerkers en biohandtekeninge ontdek vir die keuring van tuberkulose en reaksie op die behandeling. Die data is belowend vir moontlike vertaling in 'n gebruikersvriendelike toestel as 'n vinnige siftingstoets vir long-TB. Hierdie merkers en handtekeninge benodig egter verdere ondersoeke om die bruikbaarheid daarvan te verifieer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/106981
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