The presence of cytotoxic CD4 and exhausted-like CD8+ T-cells is a signature of active tuberculosis.

Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.

Flow cytometry-based method using diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease.

Authors present a pilot study of the development of innovative flow cytometry-based assay with a potential for use in tuberculosis diagnostics. Currently available tests do not provide robust discrimination between latent tuberculosis infection (TBI) and tuberculosis disease (TB). The desired application is to distinguish between the two conditions by evaluating the production of a combination of three cytokines: IL-2 (interleukin-2), IFNɣ (interferon gamma) and TNFɑ (tumor necrosis factor alpha) in CD4+ and CD8+ T cells. The study was conducted on 68 participants, divided into two arms according to age (paediatric and adults). Each arm was further split into three categories (non-infection (NI), TBI, TB) based on the immune reaction to Mycobacterium tuberculosis (M.tb) after a close contact with pulmonary TB. Each blood sample was stimulated with specific M.tb antigens present in QuantiFERON tubes (TB1 and TB2). We inferred TBI or TB based on the predominant cytokine response of the CD4+ and/or CD8+ T cells. Significant differences were detected between the NI, TBI and the TB groups in TB1 in the CD4+TNFɑ+parameter in children. Along with IL-2, TNFɑ seems to be the most promising diagnostic marker in both CD4+and CD8+ T cells. However, more detailed analyses on larger cohorts are needed to confirm the observed tendencies.

MR1-restricted T cell clonotypes are associated with "resistance" to Mycobacterium tuberculosis infection.

T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to "resist" M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ-independent T cell responses to M. tuberculosis-specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and "resistance" to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.

Mycobacterium tuberculosis is less likely to acquire pathogenic mutations during latent infection than during active disease.

Most people infected with Mycobacterium tuberculosis (Mtb) are believed to be in a state of latent tuberculosis (TB) infection (LTBI). Although LTBI is asymptomatic and not infectious, there is a risk of developing active disease even decades after infection. Here, to characterize mutations acquired during LTBI, we collected and analyzed Mtb genomes from seven Japanese patient pairs, each pair consisting of two active TB patients whose starting dates of developing active disease were >3 years apart; one had a high suspicion of LTBI before developing active disease, whereas the other did not. Thereafter, we compared these genomes with those of longitudinal sample pairs within a host of chronic active TB infections combined with public data. The bacterial populations in patients with LTBI were genetically more homogeneous and accumulated single nucleotide polymorphisms (SNPs) slower than those from active disease. Moreover, the lower proportion of nonsynonymous SNPs indicated weaker selective pressures during LTBI than active disease. Finally, the different mutation spectrums indicated different mutators between LTBI and active disease. These results suggest that the likelihood of the acquisition of mutations responsible for antibiotic resistance and increased virulence was lower in the Mtb population from LTBI than active disease.IMPORTANCEControlling latent tuberculosis (TB) infection (LTBI) activation is an effective strategy for TB elimination, where understanding Mycobacterium tuberculosis (Mtb) dynamics within the host plays an important role. Previous studies on chronic active disease reported that Mtb accumulated genomic mutations within the host, possibly resulting in acquired drug resistance and increased virulence. However, several reports suggest that fewer mutations accumulate during LTBI than during the active disease, but the associated risk is largely unknown. Here, we analyzed the genomic dynamics of Mtb within the host during LTBI. Our results statistically suggest that Mtb accumulates mutations during LTBI, but most mutations are under low selective pressures, which induce mutations responsible for drug resistance and virulence. Thus, we propose that LTBI acts as a source for new TB disease rather than as a period for in-host genome evolution.

Serum exosome tRFs as a promising biomarker for active tuberculosis and latent tuberculosis infection.

OBJECTIVE: To analyse the expression profiles of serum exosome tRFs/tiRNAs and to explore their diagnostic value in tuberculosis (TB) activity. METHODS: The serum exosome tRF/tiRNA profile was analysed using high-throughput sequencing technology in 5 active tuberculosis (ATB) patients, 5 latent tuberculosis infection (LTBI) patients and 5 healthy controls (HCs). Then, serum exosome tRFs/tiRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and their diagnostic value was evaluated by receiver operating characteristic curve (ROC) and area under the curve (AUC). Finally, bioinformatics analysis was performed to explore and identify the potential biological pathways induced by tRFs/tiRNAs. RESULTS: The sequencing results revealed that serum exosome tRF/tiRNA expression profiles were different among ATB patients, LTBI patients and HCs. Three tRFs (tRF-56:75-Trp-CCA-4, tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2) were selected for qRT-PCR validation. The results demonstrated that the expression level of tRF-1-22-chrM.Ser-GCT was upregulated in ATB patients, while tRF-56-75-Trp-CCA-4 was downregulated, which was consistent with the sequencing data. The AUCs of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM. Ser-GCT were 0.824 and 1.000, respectively, which have significant values in the diagnosis of ATB patients. Moreover, the expression levels of tRF-56:75-Trp-CCA-4 and tRF-1:22-chrM.Ser-GCT and tRF-56:76-Val-TAC-1-M2 in ATB patients and LTBI were different, which indicated that these three tRFs could effectively distinguish ATB patients and LTBI patients. CONCLUSION: Our findings indicate that serum exosome tRFs can be used as potential markers for the diagnosis of ATB and LTBI.

Whole-transcriptome sequencing of phagocytes reveals a ceRNA network contributing to natural resistance to tuberculosis infection.

Tuberculosis (TB) is a major fatal infectious disease globally, exhibiting high morbidity rates and impacting public health and other socio-economic factors. However, some individuals are resistant to TB infection and are referred to as "Resisters". Resisters remain uninfected even after exposure to high load of Mycobacterium tuberculosis (Mtb). To delineate this further, this study aimed to investigate the factors and mechanisms influencing the Mtb resistance phenotype. We assayed the phagocytic capacity of peripheral blood mononuclear cells (PBMCs) collected from Resisters, patients with latent TB infection (LTBI), and patients with active TB (ATB), following infection with fluorescent Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Phagocytosis was stronger in PBMCs from ATB patients, and comparable in LTBI patients and Resisters. Subsequently, phagocytes were isolated and subjected to whole transcriptome sequencing and small RNA sequencing to analyze transcriptional expression profiles and identify potential targets associated with the resistance phenotype. The results revealed that a total of 277 mRNAs, 589 long non-coding RNAs, 523 circular RNAs, and 35 microRNAs were differentially expressed in Resisters and LTBI patients. Further, the endogenous competitive RNA (ceRNA) network was constructed from differentially expressed genes after screening. Bioinformatics, statistical analysis, and quantitative real-time polymerase chain reaction were used for the identification and validation of potential crucial targets in the ceRNA network. As a result, we obtained a ceRNA network that contributes to the resistance phenotype. TCONS_00034796-F3, ENST00000629441-DDX43, hsa-ATAD3A_0003-CYP17A1, and XR_932996.2-CERS1 may be crucial association pairs for resistance to TB infection. Overall, this study demonstrated that the phagocytic capacity of PBMCs was not a determinant of the resistance phenotype and that some non-coding RNAs could be involved in the natural resistance to TB infection through a ceRNA mechanism.

High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL-17 against M. tuberculosis antigen.

Background: Interleukin-17-producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with human immunodeficiency virus (HIV) disproportionately affects distinct Th17-cell subsets that respond to Mtb is incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by liquid chromatography-mass spectrometry (LC/MS) on plasma and tested the hypothesis that tryptophan catabolism influences Th17-cell frequencies in this context. Results: We identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3-cells of which subset 1 was significantly reduced in latent tuberculosis infection (LTBI) with HIV-ART, yet Mtb-responsive IL-17-producing CD4 T cells were preserved; we found that IL-17-producing CD4 T cells dominate the response to Mtb antigen but not cytomegalovirus (CMV) antigen or staphylococcal enterotoxin B (SEB), and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17-cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL-17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

RATE OF INFECTION (TUBERCULOSIS) IN BRAZILIANS IBD PRIVATE PATIENTS: FOLLOW-UP 15 YEARS.

BACKGROUND: Latent tuberculosis (LTB) is a condition where the patient is infected with Mycobacterium tuberculosis but does not develop active TB. There's a possibility of tuberculosis (TB) activation following the introduction of anti-TNFs. OBJECTIVE: To assess the risk of biological therapy inducing LTB during inflammatory bowel diseases (IBD) treatment over 15 years in a high-risk area in Brazil. METHODS: A retrospective study of an IBD patients' database was carried out in a private reference clinic in Brazil. All patients underwent TST testing and chest X-ray prior to treatment, and once a year after starting it. Patients were classified according to the Montreal stratification and risk factors were considered for developing TB. RESULTS: Among the analyzed factors, age and gender were risk factors for LTB. DC (B2 and P) and UC (E2) patients showed a higher number of LTB cases with statistical significance, what was also observed for adalimumab and infliximab users, compared to other medications, and time of exposure to them favored it significantly. Other factors such as enclosed working environment have been reported as risk. CONCLUSION: The risk of biological therapy causing LTB is real, so patients with IBD should be continually monitored. This study reveals that the longer the exposure to anti-TNFs, the greater the risk. BACKGROUND: •Rate of infection (tuberculosis) in Brazilians IBD private patients: follow-up 15 years. BACKGROUND: •Patients treated with immunosuppressants and/or anti-TNFs have a higher risk of developing opportunistic infections, among them the most common is latent tuberculosis or even active tuberculosis. BACKGROUND: •Similar risks may be noted in patients with inflammatory bowel diseases (IBDs). BACKGROUND: •This study reveals that the longer the exposure to anti-TNFs, the greater the risk for de IBD patients. BACKGROUND: •The study demonstrated the importance of monitoring these patients permanently and continuously.

In vitro stimulation with nontuberculous mycobacteria induced a stronger cytokine response in leukocytes isolated from individuals with latent tuberculosis compared to those isolated from active tuberculosis or cystic fibrosis patients.

Mycobacterium tuberculosis and opportunistic environmental non-tuberculous mycobacteria (NTM) can cause severe infection. Why latent tuberculosis infection advances to active disease, and why some individuals with cystic fibrosis (CF) develop pulmonary infections with NTM is still poorly understood. The aim of this study was to investigate the effector function of peripheral blood mononuclear cells (PBMC) from individuals with active or latent tuberculosis, individuals with CF with or without pulmonary NTM-infection and healthy controls, by measuring cytokine response to in vitro stimulation with different species of NTMs. The cytokine concentrations of IL-17A, IL-22, IL-23, IL-10, IL12p70 and IFN-γ were measured in PBMC-culture supernatants after stimulation with NTMs. PBMCs from individuals with latent tuberculosis infection showed strong IL-17A, IL-22, and IFN-γ responses compared to individuals with active tuberculosis or CF. IL-10 production was low in both tuberculosis groups compared to the CF groups and controls. This study suggests that IL-17A and IL-22 might be important to keep tuberculosis in a latent phase and that individuals with CF with an ongoing NTM infection seem to have a low cytokine response.

Normalizing granuloma vasculature and matrix improves drug delivery and reduces bacterial burden in tuberculosis-infected rabbits.

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.

Comparison of the Single Cell Immune Landscape between Subjects with High Mycobacterium tuberculosis Bacillary Loads during Active Pulmonary Tuberculosis and Household Members with Latent Tuberculosis Infection.

It is unclear how the immune system controls the transition from latent tuberculosis (TB) infection (LTBI) to active pulmonary infection (PTB). Here, we applied mass spectrometry cytometry time-of-flight (CyTOF) analysis of peripheral blood mononuclear cells to compare the immunological landscapes in patients with high tuberculous bacillary load PTB infections and LTBI. A total of 32 subjects (PTB [n = 12], LTBI [n = 17], healthy volunteers [n = 3]) were included. Participants with active PTBs were phlebotomized before administering antituberculosis treatment, whereas participants with LTBI progressed to PTB at the time of household screening. In the present study, CyTOF analysis identified significantly higher percentages of mucosal-associated invariant natural killer T (MAIT NKT) cells in subjects with LTBI than in those with active PTB and healthy controls. Moreover, 6 of 17 (35%) subjects with LTBI progressed to active PTB (LTBI progression) and had higher proportions of MAIT NKT cells and early NKT cells than those without progression (LTBI non-progression). Subjects with LTBI progression also showed a tendency toward low B cell levels relative to other subject groups. In conclusion, MAIT NKT cells were substantially more prevalent in subjects with LTBI, particularly those with progression to active PTB.

Toward a molecular microbial blood test for tuberculosis infection.

The World Health Organization's aim to end the global tuberculosis (TB) epidemic by 2050 cannot be achieved without taking measures to identify people with asymptomatic Mycobacterium tuberculosis (Mtb) infection and offer them an intervention to reduce the risk of disease progression, such as preventive antimicrobial therapy. Implementation of this strategy is limited by the fact that existing tests for Mtb infection, which use immunosensitization to Mtb-specific antigens as a proxy for infection, have low positive predictive value for progression to TB. A blood test that detects Mtb deoxyribonucleic acid (DNA) could allow preventive therapy to be targeted at individuals with microbiological evidence of persistent infection. In this review, we summarize recent advances in the development of molecular microbial blood tests for Mtb infection and discuss potential explanations for discordance between their results and those of immunodiagnostic tests in adults with recent exposure to an infectious index case. We also present a roadmap for further development of molecular microbial blood tests for Mtb infection, and highlight the potential for research in this area to provide novel insights into the biology of Mtb infection and yield new tools to support efforts to control the global TB epidemic.

Using immune clusters for classifying Mycobacterium tuberculosis infection.

BACKGROUND: The differential diagnosis between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) is still a challenge worldwide. METHODS: Immune indicators involved in innate, humoral, and cellular immune cells, as well as antigen-specific cells were simultaneously assessed in patients with ATB and LTBI. RESULTS: Of 54 immune indicators, no indicator could distinguish ATB from LTBI, likely due to an obvious heterogeneity of immune indicators noticed in ATB patients. Cluster analysis of ATB patients identified three immune clusters with different severity. Cluster 1 (42.1 %) was a ''Treg/Th1/Tfh unbalance type" cluster, whereas cluster 2 (42.1 %) was an "effector type'' cluster, and cluster 3 was a ''inhibition type'' cluster (15.8 %) which showed the highest severity. A prediction model based on immune indicators was established and showed potential in classifying Mycobacterium tuberculosis infection. CONCLUSIONS: We depicted the immune landscape of patients with ATB and LTBI. Three immune subtypes were identified in ATB patients with different severity.

The potential mechanism of the progression from latent to active tuberculosis based on the intestinal microbiota alterations.

INTRODUCTION: Tuberculosis (TB) poses a serious challenge to global health systems. The altered intestinal microbiota is associated with the pathogenesis of TB, but the exact links remain unclear. METHODS: 16 S rDNA sequencing was performed to comprehensively detect the changes in the intestinal microbiota of feces from active TB (ATB), latent TB infection (LTBI) and healthy controls (HC). RESULTS: The rarefaction curves demonstrated the sequencing results' validity. The alpha diversity was lowest in ATB, while highest in HC. Boxplot of beta diversity showed significant differences in every two groups. LDA Effect Size (LEfSe) Analysis revealed differences in probiotic bacteria like Romboutsia, Bifidobacterium and Lactobacillus in LTBI, and pro-inflammatory bacteria like R. gnavus, Streptococcus and Erysipelatoclostridium in ATB, corresponding to the cluster analysis. PICRUST2 analysis revealed the pentose phosphate pathway was active in ATB and LTBI (more active in ATB). The differences between the groups are statistically significant at the P<0.05 level. CONCLUSION: Our study indicated that from LTBI to ATB, some intestinal microbiota inhibit the synthesis of interferon (INF)-γ and interleukin (IL)-17, promoting the survival and spread of Mycobacterium tuberculosis (M. tb). In addition, the metabolites secreted by intestinal microbiota and dysbiosis in intestine also have an effect on the development of LTBI to ATB.

Low performance of interferon gamma release assay Quantiferon-TB gold coupled or not with Pst1/3/lipoglycan humoral detection to predict Mycobacterium tuberculosis complex disease in a low-burden area.

Whole T cell interferon gamma release assays such as QuantiFERON-TB Gold Plus (QTF-TB) are used to evaluate Mycobacterium tuberculosis complex (MTC) exposure but fail to discriminate latent tuberculosis infection (LTBI) from active disease. In this study conducted in a low-burden area, 1215 patients presenting MTC risk and tested both for QTF-TB and mycobacterial infection (microscopy, culture, and/or PCR) were selected, as well as 1298 controls screened with QTF-TB before medical recruitment. The humoral response (LIODetect®TB-ST) was further evaluated in 199 selected patients. In patients with active disease, MTC positivity (culture and/or PCR with species identification) was associated with QTF-TB positivity (45/56, 80.4 %). Although QTF-TB1/TB2 peptides were not suitable for discriminating against active MTC disease from LTBI, the cut-off value of 4.4 IFN-γ IU/mL produced the best diagnostic performance for MTC detection. Lower levels of QTF-TB were reported among patients with isolated active pulmonary MTC as compared to a lymph-nodal location and a disseminated form. Next, antibodies were detected in 4/55 (7.3 %) active MTC disease cases, while negative in cases of LTBI and indeterminate/negative QTF-TB. In conclusion, the added value to combine cellular (QTF-TB) and humoral (LIODetect®TB-ST) assays to predict an active MTC disease is limited.

Serum proteomics analysis for differentiation among Mycobacterium tuberculosis infection categories.

Inhalation of Mycobacterium tuberculosis (Mtb) bacilli can lead to a range of TB categories including early clearance (EC), latent TB infection (LTBI) and active TB (ATB). There are few biomarkers available to differentiate among these TB categories: effective new biomarkers are badly needed. Here, we analyzed the serum proteins from 26 ATB cases, 20 LTBI cases, 34 EC cases and 38 healthy controls (HC) using label-free LC-MS/MS. The results were analyzed using MaxQuant software and matched to three different bacterial proteomics databases, including Mtb, Mycobacterium spp. and normal lung flora. PCA of protein candidates using the three proteomics databases revealed 44.5% differentiation power to differentiate among four TB categories. There were 289 proteins that showed potential for distinguishing between each pair of groups among TB categories. There were 50 candidate protein markers specifically found in ATB and LTBI but not in HC and EC groups. Decision trees using the top five candidate biomarkers (A0A1A2RWZ9, A0A1A3FMY8, A0A1A3KIY2, A0A5C7MJH5 and A0A1X0XYR3) had 92.31% accuracy to differentiate among TB categories and the accuracy was increased to 100% when using 10 candidate biomarkers. Our study shows that proteins expressed from Mycobacterium spp. have the potential to be used to differentiate among TB categories.

Latent Tuberculosis in Psoriasis Patients on Biologic Therapies: Real-World Data from a Care Center in Romania.

Background and Objectives: Psoriasis is a chronic and inflammatory condition that has a huge impact on the patient's quality of life. Biological treatment improved psoriasis therapy, with impressive results seen in the evolution of the disease and the patient's quality of life. However, the risk of mycobacterium tuberculosis (MTB) infection reactivation is well-known to biological therapy, which raises problems especially in an endemic country. Materials and Methods: In this study, we followed moderate to severe psoriasis patients who had latent tuberculosis infection (LTBI) following treatment with a biological therapy approved in Romania. Results: The patients were evaluated at baseline and then followed-up with Mantoux tests and chest X-rays every year, resulting in 54 patients being diagnosed with LTBI. At the initial evaluation, 30 patients with LTBI were identified, and 24 more were identified during biological therapy. These patients were given prophylactic treatment. Out of the 97 participants in this retrospective study, 25 required association of methotrexate (MTX) alongside biological therapy. We compared the prevalence of positive Mantoux tests in patients with combined therapy with that of patients only on biological treatment, and the results were higher in the combined therapy group. Conclusion: All the patients in the study were vaccinated against tuberculosis (TB) after birth, and none were diagnosed with active tuberculosis (aTB) before or after the start of therapy according to the pulmonologist.