Towards a better diagnosis of mycobacterial infections and beyond.

Student thesis: Doctoral thesis


The genus Mycobacterium includes over 200 species, most of them found in the environment. Mycobacterium tuberculosis, one of the species of the M. tuberculosis complex, is the causative agent of tuberculosis (TB) in humans. TB is the first cause of death worldwide due to a single infectious agent. Aditionally, 23% of the world’s population is estimated to be latently infected. Identification of these cases is key for TB control. However, diagnosis of latent tuberculosis infection (LTBI) has limitations and the existing assays cannot differentiate between LTBI and TB, differentiate between the different TB spectrum stages, or predict progression to disease. TB infection screening in patients at high risk of developing active TB is crucial. In article 1 of this thesis, IFN-γ release assays (IGRAs) were assessed in patients with immune-mediated inflammatory diseases showing that IGRAs are suitable and not affected by treatment regimens. In article 2, IGRAs and IP-10 detection were evaluated in patients with rheumatoid arthritis. IP-10 and IFN-γ detection were comparable and their combined use considered beneficial. In article 3, the impact of biological response modifiers on respiratory tract infections in these patients is reviewed. In article 4, the use of serial QuantiFERON-TB Gold In-Tube (QFN-G-IT) testing is discussed, concluding that it should be further evaluated as high IFN-γ conversion levels could indicate disease progression in children. Several approaches are studied to improve TB immunodiagnostics such as stimulating samples with other TB specific antigens, detecting other cytokines (alone or combined) (as IP-10, articles 2 and 7), and characterizing different cell populations. In article 5, adding EspC, EspF and Rv2348-B to the QFN-G-IT antigens (ESAT-6, CFP-10 and TB7.7) was evaluated. Despite a mild increase in sensitivity, such addition did not improve the performance of the current test. However, when used in the absence of ESAT-6, it yielded comparable results to the QFN-G-IT combination, offering an alternative in the case that the ESAT-6 based vaccine or tuberculin skin test (TST) are developed. In article 6, CD27 and CCR4 were evaluated on M. tuberculosis-specific CD4+ T-cells (IFN-γ+ and/or TNF-α+) using flow cytometry. In agreement with previous studies, these two markers resulted as potential TB biomarkers distinguishing active TB from LTBI patients. Article 7, focuses on addressing TB under-diagnosis by increasing the reach. IP-10 detection in dried plasma spots was evaluated in contacts and considered a good approach for LTBI screening in sites where fresh plasma samples cannot be stored and transported properly. Non-tuberculous mycobacteria (NTM) infections are increasing worldwide; however, there is no diagnostic test available to determine the clinical relevance of NTM isolates in respiratory samples. Moreover, when diagnosing TB infection, NTM can complicate interpretation by causing discordant results (negative IGRA but positive TST despite no BCG vaccination). Article 8 evaluates the use of glycopeptidolipids (GPLs, NTM-specific antigens) for a future NTM-IGRA. Stimulation with GPLs yielded higher IFN-γ levels in cases with NTM lymphadenopathies, suspicion of NTM infections and disseminated NTM infections compared to cases with active TB, LTBI and healthy controls. Regarding patients with chronic pulmonary diseases (CPD) with NTM isolates, those considered as NTM-colonized and those with a record of previous NTM-disease, had lower IFN-γ production than those classified as NTM-disease. An NTM-IGRA based on stimulation with GPLs would be a very useful tool for better handling of CPD patients with NTM isolates of unclear clinical relevance and LTBI screening cases with discordant results. Altogether, this thesis focuses on evaluating and developing different approaches beyond the current IGRAs for a better TB and NTM infection diagnosis. Adoption of improved methods would improve patient management and wellbeing and also help to decrease the TB burden.
Date of Award13 Dec 2019
Original languageSpanish
SupervisorIsidre Gibert Gonzalez (Tutor), Irene Latorre Rueda (Director), Cristina Prat Aymerich (Director) & Jose Antonio Dominguez Benitez (Director)

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