Evaluation of immunological markers for the diagnosis of active and latent tuberculosis

Tuberculosis (TB) is the single deadliest infectious disease in the world and around one fourth of the global population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). The World Health Organization (WHO) targets reduction of mortality by 95% and incidence rate by 90% by year 2035. This thesis aims to contribute by identifying novel biomarkers to distinguish the different stages of infection, from latency to active disease and thereby identify individuals in need of treatment. Currently used assays for latent TB are the tuberculin skin test (TST), which cross-reacts with other mycobacterial strains as well as with the Bacillus Calmette Guérin (BCG) vaccine, and the interferon-ɣ release assays (IGRAs) which are more specific for Mtb. However, neither assay can distinguish a previously healed from an active TB. Nor can they distinguish individuals with a recent Mtb infection with increased risk of incipient TB, from a remote and well-controlled infection. Thus, the positive predictive values for active TB are very low. There is a need for more sensitive biomarkers to identify individuals with increased risk of progression to active disease, in order to better control and prevent transmission of TB.

In the first paper FASCIA analysis with PPD and Mtb antigens was proved to be a robust assay with similar sensitivity and specificity as compared with IGRAs. The overall sensitivity for verified active TB was 86% and for latent TB 61%. FASCIA results were concordant with IGRA results in 90% of active TB cases and in 80% of individuals with LTBI. Stronger and more frequent proliferative CD4+ responses were induced in patients with extra-pulmonary TB compared to pulmonary TB (p<0.05). FASCIA performed well in patients with moderate immunosuppression. It was demonstrated in the second paper that cytokine levels were significantly higher after stimulation with CFP-10 and ESAT-6 in individuals with verified TB, compared to healthy controls (p<0.005). The chemokine IP-10 levels after stimulation with antigens CFP-10/ESAT-6 showed a significantly higher sensitivity compared to IFN-ɣ responses in individuals with active TB (p<0.05). A mathematical model was developed in the third paper using clinical and epidemiological data to estimate the probabilities of recent and remote latent TB. Results from these estimations were similar to previously published data from contact screenings for house-hold contacts after exposure to smear microscopy positive patients (35%). With a cut-off at 10% of high probability of latent TB, T-SPOT.TB detected 100% of probable recent and remote infections. A prediction model was developed in the fourth paper where the most specific markers for prediction of recent infection were early (<1 month) high proliferative CD4+ responses to CFP-10 and PPD and low responses to ESAT-6 in contacts to verified pulmonary TB. Other Mtb antigens (Rec85a, Rec85b and Rv1284) were also sensitive markers of recent infection, but did not distinguish recent from remote infection.

The findings from our studies indicate that positive predictive values for incipient TB in Mtb assays can be improved and aid clinicians in targeting those in need of treatment to prevent disease and further transmission of active TB, as well as avoid unnecessary costs and adverse events.

List of scientific papers

I. Borgström E, Andersen P, Andersson L, Julander I, Källenius G, Maeurer M, Norrby M, Rosenkrands I, Tecleab T, Bruchfeld J, Gaines H. Detection of proliferative responses to ESAT-6 and CFP-10 by FASCIA assay for diagnosis of Mycobacterium tuberculosis infection. Journal of Immunological Methods. 370 (2011) 55-64.
https://doi.org/10.1016/j.jim.2011.05.008

II. Borgström E, Andersen P, Atterfelt F, Julander I, Källenius G, Maeurer M, Rosenkrands I, Widfeldt M, Bruchfeld J, Gaines H. Immune responses to ESAT-6 and CFP-10 by FASCIA and multiplex technology for diagnosis of M. tuberculosis infection; IP-10 is a promising marker. Plos One. 2012;7(11).
https://doi.org/10.1371/journal.pone.0043438

III. Fröberg G, Wahren Borgström E, Chryssanthou E, Correia-Neves M, Källenius G, Bruchfeld J. A mathematical model to estimate the probability of recent and remote latent tuberculosis. [Manuscript]

IV. Wahren Borgström E, Fröberg G, Correia-Neves M, Atterfelt F, Bellbrant J, Szulkin R, Chryssanthou E, Ängeby K, Tecleab T, Ruhwald M, Andersen P, Källenius B, Bruchfeld J. Evaluation of proliferative CD4+ T cell responses to Mycobacterium tuberculosis antigens as predictive markers for recent infection in contacts to pulmonary tuberculosis. [Manuscript]