Suppression of host gene expression is associated with latent TB infection: a possible diagnostic biomarker.

The World Health Organization End TB strategy aims for a 90% reduction of tuberculosis (TB) incidence by 2035. Systematic testing and treatment of latent TB infection (LTBI) among contacts of active TB patients is recommended as one of the ways to curtail TB incidence. However, there is a shortage of tools to accurately diagnose LTBI. We assessed the appropriateness of whole blood host transcriptomic markers (TM) to diagnose LTBI among household contacts of bacteriologically confirmed index cases compared to HIV negative healthy controls (HC). QuantiFERON-TB Gold Plus Interferon gamma release assay (IGRA) and reverse-transcriptase quantitative PCR were used to determine LTBI and quantify TM expression respectively. Association between TM expression and LTBI was evaluated by logistic regression modelling. A total of 100 participants, 49 TB exposed (TBEx) household contacts and 51 HC, were enrolled. Twenty-five (51%) TBEx individuals tested positive by IGRA, and were denoted as LTBI individuals, and 37 (72.5%) HC were IGRA-negative. Expression of 11 evaluated TM was significantly suppressed among LTBI compared to HC. Out of the 11 TM, ZNF296 and KLF2 expression were strongly associated with LTBI and successfully differentiated LTBI from HC. Paradoxically, 21 (49%) TBEx participants who tested IGRA negative exhibited the same pattern of suppressed TM expression as IGRA positive (LTBI-confirmed individuals). Results suggest that suppression of gene expression underlies LTBI and may be a more sensitive diagnostic biomarker than standard-of-care IGRA.

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.

Interferon-Gamma Release Assay Combined with Renal Indicators to Reduce the False-Negativity of Latent Tuberculosis Infection in End-Stage Renal Disease with Hemodialysis Patients.

BACKGROUND: Tuberculosis (TB) is still the main cause of mortality due to a single transfectant, Mycobacterium tuberculosis (MTB). Latent tuberculosis infection (LTBI) is a condition characterized by the presence of tuberculosis (TB) that is not clinically apparent but nonetheless shows a sustained response to MTB. Presently, tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are mainly used to detect LTBI via cell-mediated immunity of T-cells. For people with end-stage renal disease (ESRD), the diagnosis of patients infected with MTB is difficult because of T-cell dysfunction. To get more accurate diagnosis results of LTBI, it must compensate for the deficiency of IGRA tests. METHODS: Sixty-seven hemodialysis (HD) patients and 96 non-HD patients were enrolled in this study and the study population is continuously included. IFN-γ levels were measured by the QuantiFERON-TB Gold In-Tube (QFT-GIT) test. Kidney function indicators, blood urea nitrogen (BUN), serum creatinine (Cr), and estimated glomerular filtration rate (eGFR) were used to compensate for the declined IFN-γ levels in the IGRA test. RESULTS: In individuals who were previously undetected, the results of compensation with serum Cr increased by 10.81%, allowing for about 28% more detection, and compensation with eGFR increased by 5.41%, allowing for approximately 14% more detectable potential among them and employing both of them could enhance the prior shortcomings of IGRA tests. when both are used, the maximum compensation results show a sensitivity increase rate of 8.81%, and approximately 23% of patients who were previously undetectable may be found. CONCLUSION: Therefore, the renal function markers which are routine tests for HD patients to compensate for the deficiency of IGRA tests could increase the accuracy of LTBI diagnosis.

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.

Characterization of natural killer cells in the blood and airways of cynomolgus macaques during Mycobacterium tuberculosis infection.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and kills more than 1.5 million people each year. METHODS: We examine the frequency and function of NK cells in the blood and airways over the course of Mtb infection in a TB macaque model and demonstrate differences in NK marker expression between the two compartments. Flow cytometry and intracellular cytokine staining were utilized to identify NK cell subsets (expressing NKG2A, CD56, or CD16) and function (IL-10, TNF, IL-2, IFN-g, IL-17, and CD107a). RESULTS: Blood and airway NK cell frequencies were similar during infection though there were differences in subset populations between blood and airway. Increased functional (cytokine/CD107a) parameters were observed in airway NK cells during the course of infection while none were seen in the blood. CONCLUSIONS: This study suggests that NK cells in the airway may play an important role in TB host response.

Serum levels of novel anti-inflammatory cytokine Interleukin-38 in diabetes patients infected with latent tuberculosis (DM-LTB-3).

IL-38 is a recently discovered, novel anti-inflammatory cytokine, which belongs to the IL-1ß family. The role played by this cytokine in diabetes-tuberculosis nexus is not known. Serum levels of IL-38, TNF-α, IL-6, and IL-1ß in Normal Glucose Tolerance (NGT) and chronic Diabetes (DM) subjects, both with and without latent tuberculosis (LTB) (n = 256) were quantified by ELISA. While, serum levels of IL-38 were significantly reduced, the levels of TNF-α, IL-6, and IL-1ß were not altered, in LTB infected diabetes patients. While no significant secretion of IL-38 was detected in the quantiferon supernatant, secretion of TNF-α, IL-6, and IL-1ß was significantly reduced in LTB infected diabetes patients. The decreased systemic levels of IL-38 and reduced in vitro secretion of other pro-inflammatory cytokines might represent a crucial pathway associated with diabetes-tuberculosis nexus.

Effect of SARS-CoV-2 seropositivity on antigen - specific cytokine and chemokine responses in latent tuberculosis.

SARS-CoV-2 and latent Mycobacterium tuberculosis infection are both highly co-prevalent in many parts of the globe. Whether exposure to SARS-CoV-2 influences the antigen specific immune responses in latent tuberculosis has not been investigated. We examined the baseline, mycobacterial antigen and mitogen induced cytokine and chemokine responses in latent tuberculosis (LTBI) individuals with or without SARS-CoV-2 seropositivity, LTBI negative individuals with SARS-CoV-2 seropositivity and healthy control (both LTBI and SARS-CoV-2 negative) individuals. Our results demonstrated that LTBI individuals with SARS-CoV-2 seropositivity (LTBI+/IgG +) were associated with increased levels of unstimulated and TB-antigen stimulated IFNγ, IL-2, TNFα, IL-17, IL-1ß, IL-6, IL-12, IL-4, CXCL1, CXCL9 and CXCL10 when compared to those without seropositivity (LTBI+/IgG-). In contrast, LTBI+/IgG+ individuals were associated with decreased levels of IL-5 and IL-10. No significant difference in the levels of cytokines/chemokines was observed upon mitogen stimulation between the groups. SARS-CoV-2 seropositivity was associated with enhanced unstimulated and TB-antigen stimulated but not mitogen stimulated production of cytokines and chemokines in LTBI+ compared to LTBI negative individuals. Finally, most of these significant differences were not observed when LTBI negative individuals with SARS-CoV-2 seropositivity and controls were examined. Our data clearly demonstrate that both baseline and TB - antigen induced cytokine responses are augmented in the presence of SARS-CoV-2 seropositivity, suggesting an augmenting effect of prior SARS-CoV-2 infection on the immune responses of LTBI individuals.

Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis.

Introduction: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. Materials and Methods: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. Results: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p ≤ 0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. Conclusion: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals.

CXCL17 Is Dispensable during Hypervirulent Mycobacterium tuberculosis HN878 Infection in Mice.

CXCL17 is a novel mucosal chemokine that mediates myeloid cell recruitment and bactericidal activity and highly expressed in the respiratory tract. However, its role in tuberculosis (TB) immunopathogenesis or protection remains unknown. In this study, we evaluated the function of CXCL17 in a mouse model of aerosol infection with the clinical W-Beijing lineage Mycobacterium tuberculosis hypervirulent HN878 strain. Our results show that CXCL17 production increases in the lung of M. tuberculosis-infected mice during acute and chronic stages of infection. Moreover, in vitro M. tuberculosis infection of epithelial cells and myeloid cells induces production of CXCL17. In humans, lower serum CXCL17 levels are observed among active pulmonary TB patients when compared with subjects with latent TB infection and healthy controls, suggesting a protective role. However, mice treated with rCXCL17 show similar lung bacterial burden and inflammation compared with control animals, despite an increased lung myeloid cell accumulation. Finally, CXCL17-/- mice are not more susceptible to TB than wild-type animals. These findings suggest that CXCL17 is induced in both murine epithelial and myeloid cells upon M. tuberculosis infection and increased expression during human latent TB infection. However, CXCL17 may have a dispensable role during pulmonary TB.

Distinct blood transcriptomic signature of treatment in latent tuberculosis infected individuals at risk of developing active disease.

Although only a small fraction will ever develop the active form of tuberculosis (ATB) disease, chemoprophylaxis treatment in latent TB infected (LTBI) individuals is an effective strategy to control pathogen transmission. Characterizing immune responses in LTBI upon chemoprophylactic treatment is important to facilitate treatment monitoring, and thus improve TB control strategies. Here, we studied changes in the blood transcriptome in a cohort of 42 LTBI and 8 ATB participants who received anti-TB therapy. Based on the expression of previously published gene signatures of progression to ATB, we stratified the LTBI cohort in two groups and examined if individuals deemed to be at elevated risk of developing ATB before treatment (LTBI-Risk) differed from others (LTBI-Other). We found that LTBI-Risk and LTBI-Other groups were associated with two distinct transcriptomic treatment signatures, with the LTBI-Risk signature resembling that of treated ATB patients. Notably, overlapping genes between LTBI-Risk and ATB treatment signatures were associated with risk of progression to ATB and interferon (IFN) signaling, and were selectively downregulated upon treatment in the LTBI-Risk but not the LTBI-Other group. Our results suggest that transcriptomic reprogramming following treatment of LTBI is heterogeneous and can be used to distinguish LTBI-Risk individuals from the LTBI cohort at large.

Non-IFNγ Whole Blood Cytokine Responses to Mycobacterium tuberculosis Antigens in HIV-exposed Infants.

BACKGROUND: HIV-exposed uninfected (HEU) infants have increased risk of tuberculosis (TB). Testing for Mycobacterium tuberculosis (Mtb) infection is limited by reduced Quantiferon (QFT) sensitivity in infants and tuberculin skin test (TST) cross-reactivity with Bacillus Calmette-Guérin vaccine. Our objective is to assess if non-IFNγ cytokine responses to Mtb-specific antigens have improved sensitivity in detecting Mtb infection in HEU infants compared with QFT. METHODS: HEU infants were enrolled in a randomized clinical trial of isoniazid preventive therapy (IPT) to prevent Mtb infection in Kenya (N = 300) and assessed at 12 months postrandomization (14 months of age) by TST and QFT-Plus. Non-IFNγ cytokine secretion (IL2, TNF, IP10, N = 229) in QFT-Plus supernatants was measured using Luminex assay. Logistic regression was used to assess the effect of IPT on Mtb infection outcomes in HEU infants. RESULTS: Three of 251 (1.2%) infants were QFT-Plus positive. Non-IFNγ Mtb antigen-specific responses were detected in 12 additional infants (12/229, 5.2%), all TST negative. IPT was not associated with Mtb infection defined as any Mtb antigen-specific cytokine response (odds ratio = 0.7, P = 0.54). Mtb antigen-specific IL2/IP10 responses had fair correlation (τ = 0.25). Otherwise, non-IFNγ cytokine responses had minimal correlation with QFT-Plus and no correlation with TST size. CONCLUSIONS: We detected non-IFNg Mtb antigen-specific T-cell responses in 14-month HEU infants. Non-IFNg cytokines may be more sensitive than IFNg in detecting infant Mtb infection. IPT during the first year of life was not associated with Mtb infection measured by IFNg, IL2, IP10 and TNF Mtb-specific responses.

Patterns of T and B cell responses to Mycobacterium tuberculosis membrane-associated antigens and their relationship with disease activity in rheumatoid arthritis patients with latent tuberculosis infection.

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3-6, 9-12 and 15-18 months after baseline) were performed in 41 patients who were classified empirically as 'high' (HR-T/HR-B) or 'low' (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

Microarray-based selection of a serum biomarker panel that can discriminate between latent and active pulmonary TB.

Bacterial culture of M. tuberculosis (MTB), the causative agent of tuberculosis (TB), from clinical specimens is the gold standard for laboratory diagnosis of TB, but is slow and culture-negative TB cases are common. Alternative immune-based and molecular approaches have been developed, but cannot discriminate between active TB (ATB) and latent TB (LTBI). Here, to identify biomarkers that can discriminate between ATB and LTBI/healthy individuals (HC), we profiled 116 serum samples (HC, LTBI and ATB) using a protein microarray containing 257 MTB secreted proteins, identifying 23 antibodies against MTB antigens that were present at significantly higher levels in patients with ATB than in those with LTBI and HC (Fold change > 1.2; p < 0.05). A 4-protein biomarker panel (Rv0934, Rv3881c, Rv1860 and Rv1827), optimized using SAM and ROC analysis, had a sensitivity of 67.3% and specificity of 91.2% for distinguishing ATB from LTBI, and 71.2% sensitivity and 96.3% specificity for distinguishing ATB from HC. Validation of the four candidate biomarkers in ELISA assays using 440 serum samples gave consistent results. The promising sensitivity and specificity of this biomarker panel suggest it merits further investigation for its potential as a diagnostic for discriminating between latent and active TB.

Multi-country evaluation of RISK6, a 6-gene blood transcriptomic signature, for tuberculosis diagnosis and treatment monitoring.

There is a crucial need for non-sputum-based TB tests. Here, we evaluate the performance of RISK6, a human-blood transcriptomic signature, for TB screening, triage and treatment monitoring. RISK6 performance was also compared to that of two IGRAs: one based on RD1 antigens (QuantiFERON-TB Gold Plus, QFT-P, Qiagen) and one on recombinant M. tuberculosis HBHA expressed in Mycobacterium smegmatis (IGRA-rmsHBHA). In this multicenter prospective nested case-control study conducted in Bangladesh, Georgia, Lebanon and Madagascar, adult non-immunocompromised patients with bacteriologically confirmed active pulmonary TB (ATB), latent TB infection (LTBI) and healthy donors (HD) were enrolled. ATB patients were followed-up during and after treatment. Blood RISK6 scores were assessed using quantitative real-time PCR and evaluated by area under the receiver-operating characteristic curve (ROC AUC). RISK6 performance to discriminate ATB from HD reached an AUC of 0.94 (95% CI 0.89-0.99), with 90.9% sensitivity and 87.8% specificity, thus achieving the minimal WHO target product profile for a non-sputum-based TB screening test. Besides, RISK6 yielded an AUC of 0.93 (95% CI 0.85-1) with 90.9% sensitivity and 88.5% specificity for discriminating ATB from LTBI. Moreover, RISK6 showed higher performance (AUC 0.90, 95% CI 0.85-0.94) than IGRA-rmsHBHA (AUC 0.75, 95% CI 0.69-0.82) to differentiate TB infection stages. Finally, RISK6 signature scores significantly decreased after 2 months of TB treatment and continued to decrease gradually until the end of treatment reaching scores obtained in HD. We confirmed the performance of RISK6 signature as a triage TB test and its utility for treatment monitoring.

The Frequency and Effect of Granulocytic Myeloid-Derived Suppressor Cells on Mycobacterial Survival in Patients With Tuberculosis: A Preliminary Report.

Introduction: Protective host responses in those exposed to or infected with tuberculosis (TB) is thought to require a delicate balance between pro-inflammatory and regulatory immune responses. Myeloid-derived suppressor cells (MDSCs), regulatory cells that dampen T-cell function, have been described in cancer and other infectious diseases but there are limited data on their role in TB. Methods: Peripheral blood was obtained from patients with active pulmonary TB and participants with presumed latent TB infection (LTBI) from Cape Town, South Africa. MDSC frequency was ascertained by flow cytometry. Purified MDSCs were used to assess (i) their suppressive effect on T-cell proliferation using a Ki67 flow cytometric assay and (ii) their effect on mycobacterial containment by co-culturing with H37Rv-infected monocyte-derived macrophages and autologous pre-primed effector T-cells with or without MDSCs. Mycobacterial containment was measured by plating colony forming units (CFU). Results: MDSCs (CD15+HLA-DR-CD33+) had significantly higher median frequencies (IQR) in patients with active TB (n=10) versus LTBI (n= 10) [8.2% (6.8-10.7) versus 42.2% (27-56) respectively; p=0.001]. Compared to MDSC-depleted peripheral blood mononuclear and effector T cell populations, dilutions of purified MDSCs isolated from active TB patients suppressed T-cell proliferation by up to 72% (n=6; p=0.03) and significantly subverted effector T-cell-mediated containment of H37Rv in monocyte-derived macrophages (n=7; 0.6% versus 8.5%; p=0.02). Conclusion: Collectively, these data suggest that circulating MDSCs are induced during active TB disease and can functionally suppress T-cell proliferation and subvert mycobacterial containment. These data may inform the design of vaccines and immunotherapeutic interventions against TB but further studies are required to understand the mechanisms underpinning the effects of MDSCs.

Reduced neutrophil granular proteins and post-treatment modulation in tuberculous lymphadenitis.

BACKGROUND: Neutrophils are important for host innate immune defense and mediate inflammatory responses. Pulmonary tuberculosis (PTB) is associated with increased neutrophil granular protein (NGP) levels in the circulation. However, the systemic levels of neutrophil granular proteins were not examined in tuberculous lymphadenitis (TBL) disease. METHODS: We measured the systemic levels of NGP (myeloperoxidase [MPO], elastase and proteinase 3 [PRTN3]) in TBL and compared them to latent tuberculosis (LTB) and healthy control (HC) individuals. We also measured the pre-treatment (Pre-T) and post-treatment (Post-T) systemic levels of neutrophil granular proteins in TBL individuals upon anti-tuberculosis treatment (ATT) completion. In addition, we studied the correlation and discriminatory ability of NGPs using receiver operating characteristic (ROC) analysis. RESULTS: Our data suggests that systemic levels of NGPs (MPO, PRTN3, elastase) were significantly reduced in TBL individuals compared to LTB and HC individuals. Similarly, after ATT, the plasma levels of MPO and elastase but not PRTN3 were significantly elevated compared to pre-treatment levels. NGPs (except PRTN3) were positively correlated with absolute neutrophil count of TBL, LTB and HC individuals. Further, NGPs were able to significantly discriminate TBL from LTB and HC individuals. CONCLUSION: Hence, we conclude reduced neutrophil granular protein levels might be associated with disease pathogenesis in TBL.

Mycobacterium tuberculosis-derived circulating cell-free DNA in patients with pulmonary tuberculosis and persons with latent tuberculosis infection.

OBJECTIVES: The timely diagnosis of pulmonary tuberculosis (PTB) is challenging. Although pathogen-derived circulating cell-free DNA (cfDNA) has been detected in humans, the significance of Mycobacterium tuberculosis (MTB)-cfDNA detection in patients with PTB remains unclear. METHODS: This study enrolled patients with PTB and persons with latent tuberculosis infection (LTBI) as the study and control groups, respectively, from 2018 to 2020. We measured interferon-γ levels and calculated blood monocyte-to-lymphocyte ratio (MLR). We conducted plasma cfDNA extraction, quantitative polymerase chain reaction (qPCR), and droplet digital PCR targeting the IS6110 gene of MTB. We calculated the sensitivity and specificity of using MTB-cfDNA to identify PTB and analyzed the factors associated with PTB diagnosis and MTB-cfDNA positivity. RESULTS: We enrolled 24 patients with PTB and 57 LTBI controls. The sensitivity of using MTB-cfDNA to identify PTB was 54.2%(13/24) in total and 46.2%(6/13) in smear-negative cases. Two LTBI controls (3.5%) tested positive for MTB-cfDNA, indicating a specificity of 96.5%(55/57). By using MTB-cfDNA positivity and an MLR ≥0.42 to identify PTB, sensitivity increased to 79.2%(19/24). Among patients with PTB, MTB-specific interferon-γ levels were higher in MTB-cfDNA positive participants than in those who tested negative (7.0 ±2.7 vs 2.7±3.0 IU/mL, p<0.001). MTB-cfDNA levels declined after 2 months of anti-tuberculosis therapy (p<0.001). CONCLUSION: The sensitivity of using MTB-cfDNA to identify PTB in participants was 54.2%, which increased to 79.2% after incorporating an MLR ≥0.42 into the analysis. MTB-cfDNA positivity was associated with MTB-specific immune response, and MTB-cfDNA levels declined after treatment. The clinical value of MTB-cfDNA in PTB management necessitates further investigation.

Phenotype of Peripheral NK Cells in Latent, Active, and Meningeal Tuberculosis.

The mechanisms underlying the immunopathology of tuberculous meningitis (TBM), the most severe clinical form of extrapulmonary tuberculosis (TB), are not understood. It is currently believed that the spread of Mycobacterium tuberculosis (Mtb) from the lung is an early event that occurs before the establishment of adaptive immunity. Hence, several innate immune mechanisms may participate in the containment of Mtb infection and prevent extrapulmonary disease manifestations. Natural killer (NK) cells participate in defensive processes that distinguish latent TB infection (LTBI) from active pulmonary TB (PTB). However, their role in TBM is unknown. Here, we performed a cross-sectional analysis of circulating NK cellCID="C008" value="s" phenotype in a prospective cohort of TBM patients (n = 10) using flow cytometry. Also, we addressed the responses of memory-like NK cell subpopulations to the contact with Mtb antigens in vitro. Finally, we determined plasma levels of soluble NKG2D receptor ligands in our cohort of TBM patients by enzyme-linked immunosorbent assay (ELISA). Our comparative groups consisted of individuals with LTBI (n = 11) and PTB (n = 27) patients. We found that NK cells from TBM patients showed lower absolute frequencies, higher CD69 expression, and poor expansion of the CD45RO+ memory-like subpopulation upon Mtb exposure in vitro compared to LTBI individuals. In addition, a reduction in the frequency of CD56brightCD16- NK cells characterized TBM patients but not LTBI or PTB subjects. Our study expands on earlier reports about the role of NK cells in TBM showing a reduced frequency of cytokine-producing cells compared to LTBI and PTB.

Interferon-gamma release assay levels and risk of progression to active tuberculosis: a systematic review and dose-response meta-regression analysis.

BACKGROUND: Identifying and treating individuals with high risk of progression from latent tuberculosis infection to active tuberculosis (TB) disease is critical for eliminating the disease. We aimed to conduct a systematic review and meta-regression analysis to quantify the dose-response relationship between interferon-gamma release assay (IGRA) levels and the risk of progression to active TB. METHODS: We searched PubMed and Embase from 1 January 2001 to 10 May 2020 for longitudinal studies that reported the risk of progression from latent to active TB as a function of baseline IGRA values. We used a novel Bayesian meta-regression method to pool effect sizes from included studies and generate a continuous dose-response risk curve. Our modeling framework enabled us to incorporate random effects across studies, and include data with different IGRA ranges across studies. The quality of included studies were assessed using the Newcastle-Ottawa scale (NOS). RESULTS: We included 34 studies representing 581,956 person-years of follow-up with a total of 788 incident cases of TB in the meta-regression analysis. Higher levels of interferon-gamma were associated with increased risk of progression to active tuberculosis. In the dose-response curve, the risk increased sharply between interferon-gamma levels 0 and 5 IU/ml, after which the risk continued to increase moderately but at a slower pace until reaching about 15 IU/ml where the risk levels off. Compared to 0 IU/ml, the relative risk of progression to active TB among those with interferon-gamma levels of 0.35, 1, 5, 10, 15, and 20 IU/ml were: 1.64 (1.28-2.08), 2.90 (2.02-3.88), 11.38 (6.64-16.38), 19.00 (13.08-26.90), 21.82 (14.65-32.57), and 22.31 (15.43-33.00), respectively. The dose-response relationship remains consistent when limiting the analysis to studies that scored highest in the NOS. CONCLUSION: The current practice of dichotomizing IGRA test results simplifies the TB infection disease continuum. Evaluating IGRA test results over a continuous scale could enable the identification of individuals at greatest risk of progression to active TB.

Low-dose steroids are associated with indeterminate QuantiFERON-TB Gold In-Tube assay results in immunocompetent children.

Immunocompromised status can result in indeterminate QuantiFERON-TB Gold In-Tube (QFT-GIT) results, but the association of indeterminate results with immunocompetent status in children is unknown. Therefore, we aimed to identify factors associated with indeterminate QFT-GIT results for immunocompetent children. We conducted a retrospective chart review of children (aged ≤ 18 years) who underwent QFT-GIT between September 2006 and July 2017 at the Severance Hospital, Seoul, South Korea. Of the 2037 QFT-GIT assays included in the present study, 7.7% yielded indeterminate QFT-GIT results. Multivariable logistic regression analysis identified younger age (OR 0.88; 95% CI 0.836-0.927; P < 0.001), elevated white blood cell (WBC) count (OR 1.066; 95% CI 1.020-1.115; P = 0.005), decreased albumin levels (OR 0.505; 95% CI 0.316-0.807; P = 0.004), and low-dose steroid therapy (< 1 mg/kg per day of prednisone or equivalent for < 2 weeks) (OR 76.146; 95% CI 8.940-648.569; P < 0.001) as significant factors influencing indeterminate results. Younger age, high WBC count, low albumin levels, and low-dose steroid therapy were associated with indeterminate QFT-GIT results. Low-dose steroid therapy had the highest OR for the indeterminate results compared to other significant risk factors. Our study suggests that screening for steroid doses is important prior to performing interferon-gamma release assays for immunocompetent children.