Climbing the magic mountain of tuberculosis : host related diagnostic biomarkers in blood may clear the view and get us to the top

<p dir="ltr">Tuberculosis (TB) remains a global health challenge, with an estimated 2.6 million undiagnosed cases in 2023. The performance of current microbiological diagnostic methods is inadequate particularly in extrapulmonary and paucibacillary pulmonary TB, and no reliable biomarker is available to monitor treatment response or confirm cure. This thesis investigates host-response derived biomarkers expressed in blood as diagnostic and monitoring tools for TB disease and infection.</p><p dir="ltr">In Study I and II we identified and validated three plasma protein signatures associated with TB disease. A 12-protein signature was identified in Study I through plasma profiling and network-based analysis. In Study II, the signature was refined into two condensed panels and validated in independent cohorts. The optimized 4-protein panel (CDCP1, VEGFA, IFNy, CXCL9) demonstrated the strongest discriminatory power. The overall AUC for distinguishing TB disease from TB infection was 0.86 (95% CI: 0.80-0.93) in the validation cohort. In the Italian sub cohort with only pulmonary TB, the 4-protein signature achieved an AUC of 0.92 (95% CI: 0.80-0.93) for TB disease in comparison with other respiratory diseases. All three signatures reached the minimum WHO target product profile screening test requirements in the Italian cohort, with sensitivities of 94-97% at a fixed specificity of 70%.</p><p dir="ltr">In Study III we evaluated the Xpert MTB-Host Response (MTB-HR) assay, based on a three-gene transcriptomic signature comprising an MTB-HR score [(GBP5 + DUSP3)/2 - TBP]. The study included 307 participants with presumed TB disease, out of which 103 were then diagnosed with TB disease. MTB-HR showed equal diagnostic performance in pulmonary and extrapulmonary TB (AUC 0.84 and 0.82,), with high negative predictive values (NPV 94% and 95%, respectively) at separate cut-offs. The MTB-HR score correlated strongly with bacterial burden, with lower scores observed in individuals with positive microscopy and PCR compared to Mtb culture alone. The assay was particularly effective in detecting severe disseminated forms of TB and had a higher sensitivity than sputum microscopy and PCR in pulmonary TB. MTB-HR demonstrated utility as a triage tool and screening test, with potential to reduce reliance on sputum sampling and invasive diagnostic sampling methods. Capillary and venous samples for MTB-HR were highly correlated (r = 0.97, p < . 001), allowing for a possible use as a near point-of-care test.</p><p dir="ltr">In Study IV 99 individuals were followed longitudinally during TB treatment. MTB- HR scores increased over time. A tentative reference score for cure (MTB-HRref = - 0.79) was reached by 24% of participants at four months, and some even sooner. Lower bacterial burden at baseline was associated with earlier normalization. Notably, post-treatment MTB-HR scores closely resembled those of non-TB individuals, suggesting immunologic recovery and potential utility in verifying cure. Score dynamics were more closely associated with clinical assessment of treatment response than conventional markers like CRP, indicating MTB-HR as a more specific measure of treatment effect.</p><p dir="ltr">A graded MTB-HR score may offer greater diagnostic flexibility than a single fixed threshold-especially given the wide clinical spectrum of TB disease. Establishing differentiated reference ranges and thresholds could enable Xpert MTB-HR to be used both for diagnostic, treatment monitoring and screening purposes.</p><p dir="ltr">In Study V we assessed a multiplex FluoroSpot immune assay detecting combinations of IFNy, IL2 and TNF-secreting T-cells. Triple IFNy/IL2/TNF positive cells showed superior specificity for M.tuberculosis specific immune responses. The assay revealed distinct cytokine secretion profiles across the TB spectrum. TB disease was associated with IFNy/TNF-dominant responses, while TB infection showed an IFNy/IL2 and IL2/TNF predominance. Recent contacts had higher rates of non IFNy-producing cells combined with lower cytokine secretion, suggesting early immune activation. These findings support FluoroSpot's potential to improve differentiation between TB infection stages and enhance early detection of recent TB infection.</p><p dir="ltr">In summary, these studies demonstrate that host-related biomarkers can complement existing diagnostic methods, particularly needed in extrapulmonary TB and paucibacillary pulmonary TB and for treatment monitoring. Xpert MTB-HR is a non-sputum-based diagnostic test with high negative predictive value and potential utility in screening, triage, and individualized care. The FluoroSpot assay provides functional immune profiling, could improve detection of Mtb specific immune responses and facilitate preventive treatment decisions.</p><p dir="ltr">Future directions include combining proteomic and transcriptomic signatures to enhance diagnostic precision, establishing reference ranges for Xpert MTB-HR, and exploring biomarkers for incipient TB and post-treatment cure. These approaches could transform TB diagnostics, reduce diagnostic delays, and support global TB elimination efforts.</p><h3>List of scientific papers</h3><p dir="ltr">I. A protein signature associated with active tuberculosis identified by plasma profiling and network-based analysis. Mousavian Z, <b>Folkesson E,</b> Fröberg G, Foroogh F, Correia-Neves M, Bruchfeld J, Källenius G, Sundling C. iScience. 2022 Nov 22;25(12):105652. <a href="https://doi.org/10.1016/j.isci.2022.105652" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.isci.2022.105652</a></p><p dir="ltr">II. Optimization and validation of plasma protein signatures for identification of tuberculosis disease. Mousavian Z, <b>Folkesson E,</b> Sousa Silva C, Palmieri F, Vanini V, Cuzzi G, Correia-Neves M, Goletti D, Bruchfeld J, Källenius G, Sundling S. [Submitted]</p><p dir="ltr">III. Improved Detection of Extrapulmonary and Paucibacillary Pulmonary Tuberculosis by Xpert MTB Host Response in a Tuberculosis Low-Endemic, High-Resource Setting. <b>Folkesson E,</b> Fröberg G, Sundling C, Schön T, Södersten E, Bruchfeld J. The Journal of Infectious Diseases, Volume 232, Issue 1, July 2025, Pages e78-e88. <a href="https://doi.org/10.1093/infdis/jiaf110" rel="noreferrer" target="_blank">https://doi.org/10.1093/infdis/jiaf110</a></p><p dir="ltr">IV. Xpert MTB Host Response is a promising biomarker indicating favorable treatment response in pulmonary and extrapulmonary tuberculosis. <b>Folkesson E,</b> Fröberg G, Sundling C, Zheng X, Schön T, Södersten E, Bruchfeld J. [Manuscript]</p><p dir="ltr">V. A multiplex Mtb-specific FluoroSpot assay measuring IFNy, IL2, and TNF-secreting cells can improve accuracy and differentiation across the tuberculosis spectrum. <b>Folkesson E,</b> Foroogh1 F, Kleberg L, Kjellgren V, Jakobsson M, Grunewald L, Hellberg J, Ryberg J, Maher Z, Sousa Silva C, Gower M, Grönlund H, Correia-Neves M, Makower B, Källenius G, Bruchfeld J, Sundling C. [Submitted]</p>