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Altres ajuts: This work was supported by grants from: (i) the Isolana Foundation, (ii) the Maria Francisca de Roviralta Foundation expedient AT/MA 1-19/07/2017, AT/MA 70-27/04/2016, and AT/MA 3-22/10/2014 and (vi) Fundació Recerca i Docència Mútua Terrassa. Altres ajuts: PERIS/SLT006-17-144 In recent years, pediatric research on tuberculosis (TB) has focused on addressing new biomarkers with the potential to be used as immunological non-sputum-based methods for the diagnosis of TB in children. The aim of this study was to characterize a set of cytokines and a series of individual factors (ferritin, 25-hydroxyvitamin D [25(OH)D], parasite infections, and nutritional status) to assess different patterns for discriminating between active TB and latent TB infection (LTBI) in children. The levels of 13 cytokines in QuantiFERON-TB Gold In-Tube (QFT-GIT) supernatants were analyzed in 166 children: 74 with active TB, 37 with LTBI, and 55 uninfected controls. All cytokines were quantified using Luminex or ELISA. Ferritin and 25(OH)D were also evaluated using CLIA, and Toxocara canis Ig-G antibodies were detected with a commercial ELISA kit. The combination of IP-10, IFN-γ, ferritin, and 25(OH)D achieved the best diagnostic performance to discriminate between active TB and LTBI cases in children in relation to the area under receiver operating characteristic (ROC) curve 0.955 (confidence interval 95%: 0.91-1.00), achieving optimal sensitivity and specificity for the development of a new test (93.2 and 90.0%, respectively). Children with TB showed higher ferritin levels and an inverse correlation between 25(OH)D and IFN-γ levels. The model proposed includes a combination of biomarkers for discriminating between active TB and LTBI in children to improve the accuracy of TB diagnosis in children. This combination of biomarkers might have potential for identifying the onset of primary TB in children.

El género Mycobacterium incluye más de 200 especies, la mayoría presentes en el medio ambiente. Mycobacterium tuberculosis, perteneciente al complejo M. tuberculosis, es el agente etiológico de tuberculosis (TB) en humanos. TB es la primera causa mundial de muerte por un único agente infeccioso estimándose que 23% de la población mundial está infectada. Identificar estos casos es clave para controlar la TB. Sin embargo, el diagnóstico de infección tuberculosa latente (ITBL) tiene limitaciones y las pruebas existentes no diferencian ITBL de TB, las distintas fases del espectro de TB, ni indican progresión a enfermedad. Diagnosticar infección tuberculosa en pacientes con alto riesgo de desarrollar TB es crucial. El artículo 1 de esta tesis, evalúa la detección de IFN-γ (IGRAs) en pacientes con enfermedades inflamatorias inmunomediadas mostrando los IGRAs como adecuados y no afectados por el tratamiento. El artículo 2 evalúa la detección de IFN-γ e IP-10 en pacientes con artritis reumatoide. IP-10 e IFN-γ resultaron comparables y su uso combinado considerado beneficioso. En el artículo 3, se revisa el impacto en infecciones respiratorias de modificadores de la respuesta biológica en estos pacientes. En el artículo 4, se discute el uso seriado del QuantiFERON-TB Gold In-Tube (QFN-G-IT), concluyendo que se debe evaluar, pues conversiones con altos niveles de IFN-γ podrían indicar progresión a enfermedad en niños. Para mejorar el inmunodiagnóstico de TB hay varias aproximaciones: usar otros antígenos específicos de TB, detectar otras citoquinas (solas o combinadas) (como IP-10, artículos 2 y 7), y caracterizar poblaciones celulares. El artículo 5, evalúa la adición de EspC, EspF y Rv2348-B a los antígenos del QFN-G-IT (ESAT-6, CFP-10 y TB7.7). A pesar de un ligero aumento en la sensibilidad, dicha adición no mejoró el rendimiento. Sin embargo, en ausencia de ESAT-6 se observaron resultados comparables a la combinación del QFN-G-IT, ofreciendo una alternativa al desarrollarse una vacuna o prueba de la tuberculina (PT) basadas en ESAT-6. Usando citometría, el artículo 6, analiza los marcadores CD27 y CCR4 en células T CD4+ específicas de M. tuberculosis (IFN-γ+ y/o TNF-α+) que, coincidiendo con estudios previos, resultaron potenciales biomarcadores de TB distinguiendo entre TB activa e ITBL. El artículo 7 se centra en disminuir el infradiagnóstico. Para ello, se evaluó en contactos la detección de IP-10 en gotas secas de plasma considerándose un buen método para el cribado de ITBL en lugares donde no es posible almacenar ni transportar correctamente las muestras. Las infecciones por micobacterias no tuberculosas (MNT) están aumentando. Sin embargo, determinar la relevancia clínica de MNT aisladas en muestras respiratorias no es posible actualmente. Las MNT, además, pueden complicar la interpretación de las pruebas de infección tuberculosa causando resultados discordantes (IGRA negativo, PT positiva a pesar de no estar BCG-vacunado). El artículo 8 evalúa el uso de glicopeptidolípidos (GPLs, antígenos específicos de MNT) como prueba diagnóstica de infección por MNT. Tras estimular con GPLs los niveles de IFN-γ fueron mayores en casos con linfadenopatías por MNT, sospecha de infecciones MNT y MNT diseminada, comparados con TB activa, ITBL y controles sanos. En pacientes con enfermedades pulmonares crónicas (EPC) con aislamiento de MNT, aquellos considerados como MNT-colonizados y aquellos con enfermedad por MNT previa, tuvieron menor producción de IFN-γ que aquellos clasificados como enfermos por MNT. El IGRA-MNT basado en la estimulación con GPLs sería útil para el manejo de pacientes con EPC con MNT de relevancia clínica incierta y casos con pruebas de infección tuberculosa discordantes. Esta tesis se centra en evaluar y desarrollar diferentes métodos más allá de los IGRAs para un mejor diagnóstico de infección tuberculosa y MNT, mejorando el manejo de pacientes, su bienestar y, contribuyendo a disminuir su incidencia. 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.

Background Smoking is a risk factor for tuberculosis (TB) infection and disease progression. Tobacco smoking increases susceptibility to TB in a variety of ways, one of which is due to a reduction of the IFN-gamma response. Consequently, an impaired immune response could affect performance of IFN-gamma Release Assays (IGRAs). Objective In the present study, we assess the impact of direct tobacco smoking on radiological manifestations, sputum conversion and immune response to Mycobacterium tuberculosis, analyzing IFN-gamma secretion by IGRAs. Methods A total of 525 participants were studied: (i) 175 active pulmonary TB patients and (ii) 350 individuals coming from contact tracing studies, 41 of whom were secondary TB cases. Clinical, radiological and microbiological data were collected. T-SPOT. TB and QFN-G-IT were processed according manufacturer's instructions. Results In smoking patients with active TB, QFN-G-IT (34.4%) and T-SPOT. TB (19.5%) had high frequencies of negative results. In addition, by means of an unconditional logistic regression, smoking was a main factor associated with IGRAs' false-negative results (aOR: 3.35; 95% CI: 1.47-7.61; p