Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker
| dc.contributor.author | Tsolo, Semakaleng Theresia | |
| dc.contributor.supervisor | Ranchod, Heena | |
| dc.date.accessioned | 2024-12-05T10:10:05Z | |
| dc.date.available | 2024-12-05T10:10:05Z | |
| dc.date.issued | 2023 | |
| dc.description | A research report submitted in partial fulfillment of the requirements for the degree of Master of Science (Medicine) Vaccinology to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2023 | |
| dc.description.abstract | The World Health Organization has prioritized the development of non-sputum-based assays that are capable of detecting active Tuberculosis (TB). Tryptophan (tryp) is converted to kynurenine (kyn) by the rate-limiting enzyme indoleamine 2, 3- dioxygenase (IDO). IDO activity may serve as a biomarker for active TB. Dried blood spots (DBS) can be collected outside of medical institutions and are simple to transport. We wanted to explore the use of DBS as an alternative sample type to measure the kyn/tryp ratio and IDO mRNA gene expression in healthy people. METHODS We optimised methods for elution of dried blood spots, exploring various elution buffers. Following method optimisation, we enrolled 40 healthy participants, and collected whole blood and DBS samples. Kyn and tryp concentrations were measured using ELISA (ImmuSmol, France). IDO mRNA gene expression was determined by real-time PCR using two housekeeping genes GAPDH and BACT. Statistical analysis was performed to determine the correlation and agreement between peripheral blood samples and DBS. RESULTS For IDO activity, tryp showed good agreement between plasma and DBS with a median percentage similarity of 91.1%. In contrast, no agreement was observed for kyn with a median percentage similarity of 56.6%. The kyn/tryp ratio performed poorly due to poor detection of kyn in DBS. Percentage similarity between whole blood and DBS IDO mRNA expression using GAPDH 87.1%, while using BACT was 84.6%. Using either traditional sample types or DBS, there was no correlation between IDO gene expression and kyn/tryp ratio. CONCLUSION We showed that tryp was measurable in DBS. Tryp in DBS was 91.1% similar to values in plasma. Despite method optimization, there was poor agreement between DBS and plasma for kyn. Although IDO mRNA gene expression was detectable in DBS, method agreement with whole blood was unsatisfactory. Alternative methods for the stabilization of kyn in DBS should be explored in future studies. IDO mRNA expression should be measured from whole blood in future studies | |
| dc.description.submitter | MM2024 | |
| dc.faculty | Faculty of Health Sciences | |
| dc.identifier.citation | Tsolo, Semakaleng Theresia. (2024). Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker [Master’s dissertation, University of the Witwatersrand, Johannesburg]. WireDSpace. | |
| dc.identifier.uri | https://hdl.handle.net/10539/43144 | |
| dc.language.iso | en | |
| dc.publisher | University of the Witwatersrand, Johannesburg | |
| dc.rights | © 2023 University of the Witwatersrand, Johannesburg. All rights reserved. The copyright in this work vests in the University of the Witwatersrand, Johannesburg. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of University of the Witwatersrand, Johannesburg. | |
| dc.rights.holder | University of the Witwatersrand, Johannesburg | |
| dc.school | School of Pathology | |
| dc.subject | Tuberculosis (TB) | |
| dc.subject | Indoleamine 2, 3- dioxygenase (IDO) | |
| dc.subject | UCTD | |
| dc.subject.other | SDG-3: Good health and well-being | |
| dc.title | Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker | |
| dc.type | Dissertation |