Measurement of indoleamine 2, 3-dioxygenase activity, a potential biomarker for active tuberculosis disease in South Africa

dc.contributor.authorAdu-Gyamfi, Clement
dc.date.accessioned2021-11-23T12:35:25Z
dc.date.available2021-11-23T12:35:25Z
dc.date.issued2021
dc.descriptionA thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2021en_ZA
dc.description.abstractThe Global Action Plan to End Tuberculosis (TB) has set out a 90-90-90 strategy, aiming to reach at least 90% of people with active TB, place at least 90% of them on appropriate treatment and ensure that 90% of those on treatment complete their treatment course. A key intervention to achieve this goal is the validation of a non-sputum-based diagnostic TB biomarker that can detect all forms of active TB. TB biomarker studies have been ongoing for decades. HIV is a major risk factor for TB, but previous TB biomarkers have not performed well in HIV-infected patients. Recently, the activity of indoleamine 2,3-dioxygenase 1 (IDO), has attracted attention as a TB biomarker. IDO converts tryptophan (T), an essential amino acid, to kynurenine (K). In TB, IDO directs the host immune response to Mycobacterium Tuberculosis (Mtb) towards an anti-inflammatory profile. In the first section of this work, we reviewed the literature regarding IDO in TB disease as well as in HIV infection. Broad evidence available indicates that IDO-mediated tryptophan catabolism via the kynurenine-nicotinamide pathway represents a common axis upregulated in TB and HIV-infection. Evidence spanning over three decades suggests that elevated IDO activity (high K/T ratio) is a key factor marking progression from latent TB infection to active disease, particularly in HIV-infected individuals. The gold standard method for detecting IDO activity is the measurement of the product (kynurenine)-to-substrate (tryptophan) (K/T) ratio using mass spectrometry, a time-consuming and costly procedure. In the second part of this study, we evaluated an enzyme-linked immunosorbent assay (ELISA) as an alternate method for plasma K/T ratio quantification. ELISA is a low-cost, high-throughput method, with the potential for automation, making it easy to use in peripheral laboratories. The assay requires small sample volumes and can be completed within a relatively short time. We found strong reproducibility and good agreement between plasma K/T ratio measured by ELISA and mass spectrometry. The mean bias between ELISA and mass spectrometry was 0.01 with 95% limits of agreement from -0.06 to 0.10. We then used the ELISA method to determine plasma K/T ratio in two nested case-control studies, aiming to test whether plasma IDO activity measured by K/T ratio meets the proposed target product profile of a non-sputum-based TB biomarker. In the first study, we used residual plasma samples from HIV-infected and uninfected individuals with and without active TB from Cape Town. We found that HIV-infected patients with active TB had higher plasma K/T ratio than those without TB (median 0.101 [interquartile ranges (IQR) 0.091-0.140] versus 0.061 [IQR 0.034-0.077], p < 0.0001). At a cut-off value of 0.080, plasma K/T ratio gave a sensitivity of 90%, specificity of 80%, a positive predictive value (PPV) of 82% and a negative predictive value (NPV) of 90%. In a receiver operating characteristic (ROC) analysis, plasma K/T ratio had an area under the curve (AUC) of 0.93. In HIV-uninfected patients, we also observed a higher plasma K/T ratio in those with active TB compared to those with latent TB infection (median 0.064 [IQR 0.040-0.088] versus 0.022 [IQR 0.016-0.027], p < 0.0001). A cut-off of 0.040 gave a sensitivity of 85%, a specificity of 92%, a PPV of 91% and an NPV of 84% with an AUC of 0.93. In the third section of this work, we validated our findings using the Tshepiso HIV-infected pregnant women cohort and explored whether pregnancy is a confounder to the use of plasma K/T ratio as a diagnostic biomarker for active TB. We found that plasma K/T ratio was significantly elevated during pregnancy compared with non-pregnant periods (p < 0.0001). At the time of TB diagnosis, plasma K/T ratio was markedly elevated in pregnant patients with active TB compared to individuals without TB (p < 0·0001). Applying a previously optimised cut-off value of 0.080, plasma K/T ratio gave a diagnostic sensitivity of 96%, specificity of 86%, a PPV of 77% and an NPV of 99%. A ROC gave an area under the curve of 0.95. The optimal cut-off in HIV-infected pregnant women was however, calculated as 0.100 with a sensitivity of 94%, specificity of 90%, a PPV of 85% and an NPV of 98%. Thus, we assert that the higher cut-off is more appropriate in pregnancy. A novel finding from this work was that in our control group, pregnant women who received isoniazid preventative treatment (IPT) had lower plasma K/T ratio than those who did not receive IPT, suggesting that isoniazid alone decreases IDO activity. Additionally, in both cohorts, we noted that plasma K/T ratio inversely correlated with body mass index (BMI) in TB patients, but not in controls. Relative loss in weight is a cardinal symptom of TB; thus, our findings suggest that elevated K/T ratio accompanies lower BMI as a factor associated with active TB. Conclusion In conclusion, plasma K/T ratio is a suitable blood-based biomarker of active TB in both HIV infected and uninfected individuals. Plasma K/T ratio can be used as an initial blood-based test for active TB. Pregnancy was not a confounder to the use of plasma K/T ratio to diagnose active TB in HIV infected individuals, although a higher cut-off value of 0.100 rather than 0.080 was preferable. Plasma K/T ratio can be measured using a high-throughput, low-cost, widely available ELISA. Plasma K/T ratio should be evaluated prospectively in further studies to determine its impact on patient careen_ZA
dc.description.librarianTL (2021)en_ZA
dc.facultyFaculty of Health Sciencesen_ZA
dc.identifier.urihttps://hdl.handle.net/10539/32048
dc.language.isoenen_ZA
dc.phd.titlePHDen_ZA
dc.schoolSchool of Pathologyen_ZA
dc.titleMeasurement of indoleamine 2, 3-dioxygenase activity, a potential biomarker for active tuberculosis disease in South Africaen_ZA
dc.typeThesisen_ZA
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