Evaluation of host transcriptional biomarkers in the diagnosis of TB disease and optimization of multi-colour immunophenotyping assay from the QuantiFERON® TB GOLD Plus cell sediments
| dc.contributor.advisor | Chegou, Novel N. | en_ZA |
| dc.contributor.advisor | Gutschmidt, Andrea | en_ZA |
| dc.contributor.author | Ndou, Sedzani | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. | en_ZA |
| dc.date.accessioned | 2020-11-30T10:21:07Z | en_ZA |
| dc.date.accessioned | 2021-02-01T07:56:25Z | en_ZA |
| dc.date.available | 2020-11-30T10:21:07Z | en_ZA |
| dc.date.available | 2021-02-01T07:56:25Z | en_ZA |
| dc.date.issued | 2020-11 | en_ZA |
| dc.description | Thesis (MSc)--Stellenbosch University, 2023. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Background QuantiFERON-TB Gold-Plus (QFT-Plus) test are designed for the diagnosis of Mycobacterium tuberculosis (M. tb) sensitization, hence it’s low specificity in the diagnosis of active TB. It remains unknown if the cell sediments left-over after performing the QFT-Plus ELISA can be useful for the diagnosis of active TB. Objectives To assess the expression of selected genes that have been shown in the literature as TB diagnostic or prognostic candidate biomarkers, in the cell sediments left-over after performance of the QFT-Plus test, and ascertain whether such genes when detected in QFT-Plus sediments, have any potential in the diagnosis of active TB. Methods We analysed the expression level of 13 genes namely: STAT1, GBP1, GBP5, TRAFD1, SERPING1, DUSP3, BATF2, TAP1, ETV7, SCARF1, FCGR1B, KLF2 and, GBP2, from the QFT-Plus left-over sediments using Taqman-probe qRT-PCR arrays in a total of 44 patients who were enrolled after presenting with the signs and symptoms of TB. The diagnostic accuracy of genes was evaluated using receiver operator characteristics (ROC) curves analysis whereas multi-biomarker analysis techniques were used to assess the usefulness of combinations between different genes in the diagnosis of TB. Some of the sediments from the QFT-Plus were fixed and stored for Flow Cytometry analysis in the future. The Flow Cytomery panel was designed to include the T cell subsets as well as other immune biomarkers such as IL-1B, CX3CR1, CD154 and CD56 which have been previously shown to have potential in the diagnosis of TB disease using Luminex platform Results Of the 13 individual genes evaluated in this study, the expression of 5 genes namely, BATF2, KLF2, DUSP3, GBP1 and GBP5 was significantly different (P<0.05) between the TB patients and individuals with other respiratory diseases (ORD). Individually, these genes diagnosed TB with area under the ROC curve (AUC) ≥0.89 in both the unstimulated and M. tb antigen-stimulated samples. When the genes detected in the unstimulated (Nil) sediments were fitted into general discriminant analysis (GDA) models, a 3-marker biosignature consisting of BATF2, GBP1 and GBP5 was found to be the most optimal in diagnosing TB and performed with an AUC of 0.99. However, when the Nil-derived 3-marker signature was applied on TB1 stimulated samples, the AUC reduced slightly to 0.98 and when applied to TB2 stimulated samples, the AUC remained the same as what was observed for unstimulated samples. Overall, stimulation of blood with different antigens led to different expression patterns for different genes in the specimens. Conclusion In conclusion, the results presented in this thesis show that sediments left-over after supernatants are harvested for performance of the QFT-Plus test, and which are often discarded, may be valuable samples for evaluation of both unspecific and M. tb antigen-specific gene expression. We furthermore showed that the detection of such genes may be a valuable method for the diagnosis of active TB and for future implementation purposes especially where limited blood volumes are available. Our sample size was small; hence these findings require further validation in larger studies. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Agtergrond QuantiFERON-TB Gold-Plus (QFT-Plus) -toets is ontwerp vir die diagnose van Mycobacterium tuberculosis (M. tb) -sensitisering, en daarom is dit 'n lae spesifisiteit in die diagnose van aktiewe TB. Dit bly onbekend of die oorblyfsels van die sel na die uitvoering van die QFT-Plus ELISA nuttig kan wees vir die diagnose van aktiewe TB. Doelstellings Om die uitdrukking van geselekteerde gene wat in die literatuur as TB-diagnostiese of prognostiese kandidaat-biomerkers aangetoon is, te beoordeel, in die oorblyfsels van die sel na die uitvoering van die QuantiFERON-TB Gold-Plus (QFT-Plus-toets, en om vas te stel of sulke gene in QFT-Plus-sedimente opgespoor word , het enige potensiaal in die diagnose van aktiewe TB. Metodes Ons het die uitdrukkingsvlak van 13 gene geanaliseer, naamlik: STAT1, GBP1, GBP5, TRAFD1, SERPING1, DUSP3, BATF2, TAP1, ETV7, SCARF1, FCGR1B, KLF2 en, GBP2, uit die QFT-Plus oorblywende sedimente met behulp van Taqman-sonde. qRT-PCR-skikkings by altesaam 44 pasiënte wat ingeskryf is nadat hulle die tekens en simptome van TB aangebied het. Die diagnostiese akkuraatheid van gene is geëvalueer met behulp van ROC-kromme-analise van ontvangeroperateur, terwyl multibiomerkerontledingstegnieke gebruik is om die bruikbaarheid van kombinasies tussen verskillende gene in die diagnose van TB te bepaal. Sommige van die sedimente van die QFT-Plus is in die toekoms vasgestel en gestoor vir vloei-sitometrie-analise. Die Flow Cytomery-paneel is ontwerp om die onderliggende T-sel sowel as ander immuun-biomerkers soos IL-1B, CX3CR1, CD154 en CD56 in te sluit, wat voorheen getoon is dat hulle potensiaal het in die diagnose van TB-siekte met behulp van Luminex-platform. Resultate Van die 13 individuele gene wat in hierdie studie geëvalueer is, was die uitdrukking van 5 gene, naamlik BATF2, KLF2, DUSP3, GBP1 en GBP5, betekenisvol verskillend (P <0,05) tussen TB-pasiënte en individue met ander respiratoriese siektes (ORD). Individueel het hierdie gene TB met oppervlakte onder die ROC-kurwe (AUC) ≥0,89 gediagnoseer in beide die ongestimuleerde en M. tb antigeen-gestimuleerde monsters. Toe die gene wat in die onge-gestimuleerde (Nul) sedimente opgespoor is, in algemene diskriminerende analise (GDA) -modelle toegepas is, is 'n 3-merker-biosignatuur bestaande uit BATF2, GBP1 en GBP5 gevind dat dit die beste is om TB te diagnoseer en uitgevoer met 'n AUC van 0,99. Wanneer die Nil-afgeleide 3-merkerhandtekening egter op TB1-gestimuleerde monsters aangebring is, het die AUC effens verminder tot 0,98 en wanneer dit op TB2-gestimuleerde monsters toegepas is, het die AUC dieselfde gebly as wat waargeneem is vir ongestimuleerde monsters. Oor die algemeen het stimulasie van bloed met verskillende antigene gelei tot verskillende uitdrukkingspatrone vir verskillende gene in die monsters. Afsluiting Ter afsluiting, die resultate wat in hierdie tesis aangebied word, toon aan dat die oorblyfsels van die oorblyfsels nadat die supernatante geoes is vir die uitvoering van die QFT-Plus-toets, en wat dikwels weggegooi word, waardevolle monsters kan wees vir die evaluering van onspesifieke en M. tb-antigeen- spesifieke geenuitdrukking. Verder het ons getoon dat die opsporing van sulke gene 'n waardevolle metode kan wees vir die diagnose van aktiewe TB en vir toekomstige implementeringsdoeleindes, veral waar beperkte bloedvolumes beskikbaar is. Ons steekproefgrootte was klein; hierdie bevindings vereis dus verdere validering in groter studies. | af_ZA |
| dc.description.version | Masters | en_ZA |
| dc.embargo.terms | 2023-01-01 | af_ZA |
| dc.format.extent | xviii, 144 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/109467 | af_ZA |
| dc.language.iso | en_ZA | en_ZA |
| dc.subject.lcsh | Biochemical markers | en_ZA |
| dc.subject.lcsh | Tuberculosis -- Diagnosis | en_ZA |
| dc.subject.lcsh | Gene expression | en_ZA |
| dc.subject.lcsh | Flow cytometry -- Diagnostic use | en_ZA |
| dc.subject.lcsh | UCTD | en_ZA |
| dc.title | Evaluation of host transcriptional biomarkers in the diagnosis of TB disease and optimization of multi-colour immunophenotyping assay from the QuantiFERON® TB GOLD Plus cell sediments | en_ZA |
| dc.type | Thesis | en_ZA |