Active Tuberculosis Group Research Articles

Alterations in purine and pyrimidine metabolism associated with latent tuberculosis infection: insights from gut microbiome and metabolomics analyses.

Individuals with latent tuberculosis infection (LTBI) account for almost 30% of the population worldwide and have the potential to develop active tuberculosis (ATB). Despite this, the current understanding of the pathogenesis of LTBI is limited. The gut microbiome can be altered in tuberculosis patients, and an understanding of the changes associated with the progression from good health to LTBI to ATB can provide novel perspectives for understanding the pathogenesis of LTBI by identifying microbial and molecular biomarkers associated therewith. In this study, fecal samples from healthy controls (HC), individuals with LTBI and ATB patients were collected for gut microbiome and metabolomics analyses. Compared to HC and LTBI subjects, participants with ATB showed a significant decrease in gut bacterial α-diversity. Additionally, there were significant differences in gut microbial communities and metabolism among the HC, LTBI, and ATB groups. PICRUSt2 analysis revealed that microbiota metabolic pathways involving the degradation of purine and pyrimidine metabolites were upregulated in LTBI and ATB individuals relative to HCs. Metabolomic profiling similarly revealed that purine and pyrimidine metabolite levels were decreased in LTBI and ATB samples relative to those from HCs. Further correlation analyses revealed that the levels of purine and pyrimidine metabolites were negatively correlated with those of gut microbial genera represented by Ruminococcus_gnavus_group (R. gnavus), and the levels of R. gnavus were also positively correlated with adenosine nucleotide degradation II, which is a purine degradation pathway. Moreover, a combined signature including hypoxanthine and xanthine was found to effectively distinguish between LTBI and HC samples (area under the curve [AUC] of training set = 0.796; AUC of testing set = 0.924). Therefore, through gut microbiome and metabolomic analyses, these findings provide valuable clues regarding how alterations in gut purine and pyrimidine metabolism are linked to the pathogenesis of LTBI.IMPORTANCEThis study provides valuable insight into alterations in the gut microbiome and metabolomic profiles in a cohort of adults with LTBI and ATB. Perturbed gut purine and pyrimidine metabolism in LTBI was associated with the compositional alterations of gut microbiota, which may be an impetus for developing novel diagnostic strategies and interventions targeting LTBI.

HBHA induces IL-10 from CD4+ T cells in patients with active tuberculosis but IFN-γ and IL-17 from individuals with Mycobacterium tuberculosis infection.

To effectively control tuberculosis (TB), it is crucial to distinguish between active TB disease and latent TB infection (LTBI) to provide appropriate treatment. However, no such tests are currently available. Immune responses associated with active TB and LTBI are dynamic and exhibit distinct patterns. Comparing these differences is crucial for developing new diagnostic methods and understanding the etiology of TB. This study aimed to investigate the relationship between pro- and anti-inflammatory CD4+ cytokine production following stimulation with two types of latency-associated Mycobacterium tuberculosis (M.tb) antigens to allow differentiation between active TB and LTBI. Cryopreserved PBMCs from patients with active TB disease or LTBI were stimulated overnight with replication-related antigen [ESAT-6/CFP-10 (E/C)] or two latency-associated antigens [heparin-binding hemagglutinin (HBHA) and alpha-crystallin-like protein (Acr)]. Responses were analyzed using multiparameter flow cytometry: active TB disease (n=15), LTBI (n=15) and ELISA: active TB disease (n=26) or LTBI (n=27). CD4+ central memory T cells (Tcm) specific to E/C and CD4+ effector memory T cells specific to Acr and HBHA were higher in LTBI than in TB patients. IFN-γ+Tcm and IL-17+ Tem cells was higher in the LTBI group (p= 0.012 and p=0.029 respectively), but IL-10+ Tcm was higher in the active TB group (p= 0.029) following HBHA stimulation. Additionally, following stimulation with HBHA, IL-10 production from CD4+ T cells was significantly elevated in patients with active TB compared to those with LTBI (p= 0.0038), while CD4+ T cell production of IL-17 and IFN-γ was significantly elevated in LTBI compared to active TB (p= 0.0076, p< 0.0001, respectively). HBHA also induced more CCR6+IL-17+CD4Tcells and IL-17+FoxP3+CD25+CD4Tcells in LTBI than in TB patients (P=0.026 and P=0.04, respectively). HBHA also induced higher levels of IFN-γ+IL-10+CD4+ T cells in patients with active TB (Pp=0.03) and higher levels of IFN-γ+IL-17+ CD4+ T cells in those with LTBI (p=0.04). HBHA-specific cytokine production measured using ELISA showed higher levels of IFN-γ in participants with LTBI (P=0.004) and higher levels of IL-10 in those with active TB (P=0.04). Stimulation with HBHA and measurement of CD4+ T cell production of IFN-γ, IL-10, and IL-17 could potentially differentiate active TB from LTBI. The characteristics of cytokine-expressing cells induced by HBHA also differed between participants with active TB and LTBI.

Specific Cytokines Analysis Incorporating Latency-Associated Antigens Differentiates Mycobacterium tuberculosis Infection Status: An Exploratory Study.

Current immunologic methods cannot distinguish Mycobacterium tuberculosis (Mtb) infection statuses, especially to discriminate active tuberculosis (ATB) from latent tuberculosis infection (LTBI). This study explored the potential of latency-associated antigens (Rv1733cSLP and Rv2028c) and multifactorial cytokine detection to distinguish tuberculosis infection states. ATB patients (20), LTBI healthcare workers (25), fever patients (11), and healthy controls (10) were enrolled. Cytokine levels (IFN-γ, TNF-α, IL-2, IL-6, IP-10, IL-1Ra, CXCL-1, and MCP-1) were measured using Luminex with/without MTB-specific virulence factor and latency-associated antigens stimulation. Without antigen stimulation, IL-6, IP-10, MCP-1, and IL-1Ra were higher in the ATB group than in the LTBI group (p<0.05), but no significant differences between the ATB group and the fever group. Stimulated with the four antigens, respectively, the cytokines, including IP-10Esat-6, IP-10CFP-10, IFN-γRv1733cSLP, IFN-γRv2028c, IL-6Esat-6, IL-6Rv1733cSLP, IL-6Rv2028c, IL-2Rv1733cSLP, IL-2 Rv2028c, IL-1RaEsat-6, IL-1RaCFP-10, IL-1RaRv2028c, CXCL-1Esat-6, CXCL-1CFP-10, CXCL-1Rv1733cSLP, CXCL-1Rv2028c, MCP-1Esat-6 and MCP-1CFP-10, demonstrated accurate discrimination between ATB and LTBI (p<0.05). Additive concentrations demonstrated significant secretion differences of IFN-γ, IP-10 and IL-2, primarily by virulence factors in ATB and latency-associated antigens in LTBI. Latency-associated antigens synergized with virulence factors, enhancing TH1-type cytokine diagnostic efficacy for discriminating ATB from LTBI, the AUC for TNF-α increased from 0.696 to 0.820 (p=0.038), IFN-γ increased from 0.806 to 0.962 (p=0.025), and IL-2 increased from 0.565 to 0.868 (p=0.007). Model selected by forward likelihood method indicated combined detection of IFN-γCFP-10, IFN-γRv1733cSLP, IP-10Rv1733cSLP, and CXCL-1Rv1733cSLP achieved ATB diagnosis (AUC=0.996) and ATB-LTBI differentiation (AUC=0.992). Combined detection of IFN-γCFP-10 and IFN-γRv1733cSLP achieved tuberculosis infection diagnosis (AUC=0.943). Latency-associated antigens enhance multiple cytokine discriminatory ability, particularly TH1-type cytokines, for differentiating Mtb infection statuses.

The changes and its significance of peripheral blood NK cells in patients with tuberculous meningitis.

Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). The purpose of this study was to explore the relationship between the number of natural killer (NK) cells and adaptive immune status, and disease severity in TBM patients. We conducted a retrospective study on 244 TB patients and 146 healthy control subjects in the 8th Medical Center of the PLA General Hospital from March 2018 and August 2023. The absolute count of NK cells in the peripheral blood of TBM patients was significantly lower than that in normal controls (NC), latent tuberculosis infection (LTBI), and non-severe TB (NSTB) patients (p < 0.05). The proportion of TBM patients (48.7%) with a lower absolute count of NK cells than the normal reference value was significantly higher than that in NC (5.2%) and LTBI groups (4.0%) (p < 0.05), and slightly higher than that in NSTB group (36.0%) (p > 0.05). The absolute counts of lymphocyte subsets in TBM combined with other active TB group, etiology (+) group, IGRA (-) group, and antibody (+) group were lower than that in simple TBM group, etiology (-) group, IGRA (+) group, and antibody (-) group, respectively. The CD3+ T, NK, and B cells in BMRC-stage III TBM patients were significantly lower than those in stage I and stage II patients (p < 0.05). The counts of CD3+ T, CD4+ T, and B cells in the etiology (+) group were significantly lower than those in the etiology (-) group (p < 0.05). The absolute counts of lymphocyte subsets in the peripheral blood of TBM patients were significantly decreased, especially in NK cells. The reduction of these immune cells was closely related to the disease severity and had a certain correlation with cellular and humoral immune responses. This study helps to better understand the immune mechanism of TBM and provides reliable indicators for evaluating the immune status of TBM patients in clinical practice.

Multiple cytokine analysis based on QuantiFERON-TB gold plus in different tuberculosis infection status: an exploratory study

BackgroundMore efficient and convenient diagnostic method is a desperate need to reduce the burden of tuberculosis (TB). This study explores the multiple cytokines secretion based on QuantiFERON-TB Gold Plus (QFT-Plus), and screens for optimal cytokines with diagnostic potential to differentiate TB infection status.MethodsTwenty active tuberculosis (ATB) patients, fifteen patients with latent TB infection (LTBI), ten patients with previous TB and ten healthy controls (HC) were enrolled. Whole blood samples were collected and stimulated by QFT-Plus TB1 and TB2 antigens. The levels of IFN-γ, TNF-α, IL-2, IL-6, IL-5, IL-10, IP-10, IL-1Ra, CXCL-1 and MCP-1 in supernatant were measured by Luminex bead-based multiplex assays. The receiver operating characteristic curve was used to evaluate the diagnostic accuracy of cytokine for distinguishing different TB infection status.ResultsAfter stimulation with QFT-Plus TB1 and TB2 antigens, the levels of all cytokines, except IL-5 in TB2 tube, in ATB group were significantly higher than that in HC group. The levels of IL-1Ra concurrently showed the equally highest AUC for distinguishing TB infection from HC, followed by the levels of IP-10 in both TB1 tube and TB2 tube. Moreover, IP-10 levels displayed the largest AUC for distinguishing ATB patients from non-ATB patients. Meanwhile, the levels of IP-10 also demonstrated the largest AUC in both TB1 tube and TB2 tube for distinguishing ATB patients from LTBI.ConclusionsIn addition to conventional detection of IFN-γ, measuring IP-10 and IL-1Ra based on QFT-Plus may have the more tremendous potential to discriminate different TB infection status.

Diagnostic Efficiency of the Blood-Based Cepheid 3-Gene Host Response Test and Urine-Based Lipoarabinomannan for Active Tuberculosis Case Detection at a General Hospital in China.

To assess the diagnostic performance of the blood-based Cepheid 3-gene Host Response test (MTB-HR), urine-based Lipoarabinomannan (LAM), and a combination of MTB-HR and LAM (MTB-HR & LAM) for detecting active tuberculosis (ATB). All participants were recruited from the First Affiliated Hospital, Zhejiang University School of Medicine, between June 8, 2023 and September 13, 2023. Subsequently, the participants were classified into the ATB group or non-active tuberculosis (non-ATB) group based on microbiological evidence. MTB-HR and LAM tests were performed using fingerstick blood and urine samples from each participant, respectively. The diagnostic performance of the tests was evaluated based on the sensitivity, specificity, Youden index, and Kappa value. Pairwise comparisons of the areas under the receiver operating characteristic curves (AUROCs) between different tests were conducted using nonparametric methods. A total of 297 participants were included. The MTB-HR test demonstrated diagnostic efficacy with a sensitivity of 77.37% (95% CI: 70.37-84.38) and a specificity of 85.63% (95% CI: 80.19-91.06). The LAM test demonstrated a high specificity of 97.50% (95% CI: 95.08-99.92), albeit with a lower sensitivity of 54.74% (95% CI: 46.41-63.082). The sensitivity and specificity of the MTB-HR & LAM were 83.21% (95% CI: 76.95-89.47) and 83.13% (95% CI: 77.32-88.93), respectively. Only MTB-HR & LAM exhibited higher values of area under the receiver operating characteristic curve than the LAM test (MTB-HR & LAM vs LAM: 0.83 vs 0.76, P=0.0031). In this study, although both non-sputum-based triage MTB-HR and LAM do not meet the WHO diagnostic target currently, they show possible values for triage and diagnosis in ATB. Compared to single MTB-HR or LAM test, the combined MTB-HR & LAM does not demonstrate advantages.

Alterations in the intestinal microbiota associated with active tuberculosis and latent tuberculosis infection

ObjectivesTo study the characteristics of intestinal microbiota at different stages of Mycobacterium tuberculosis infection. MethodsFecal samples of 19 active tuberculosis (ATB) patients, 21 latent tuberculosis infection (LTBI) individuals, and 20 healthy controls (HC) were collected. Gut microbiota of all the participants were analyzed by 16S rDNA sequencing. Clinical information of ATB patients was also collected and analyzed. ResultsBoth ATB and LTBI groups showed significant decreases in microbial diversity and decline of Clostridia. For ATB patients, bacteria within phylum Proteobacteria increased. While for LTBI individuals, genera Prevotella and Rosburia enriched. The abundance of Faecalibacterium, Clostridia and Gammaproteobacteria has the potential to diagnose ATB, with the area under the curve (AUC) of 0.808, 0.784 and 0.717. And Prevotella and Rosburia has the potential to diagnose LTBI, with the AUC of 0.689 and 0.689. Notably, in ATB patients, the relative abundance of Blautia was negatively correlated with the proportions of peripheral T cells and CD8+T cells. And serum direct bilirubin was positively correlated with Bacteroidales, while negatively correlated with Clostridiales in ATB patients. ConclusionsThe specifically changed bacteria are promising markers for ATB and LTBI diagnosis. Some gut bacteria contribute to anti-MTB immunity through interactions with T cells and bilirubin.

A novel chemokine biomarker to distinguish active tuberculosis from latent tuberculosis: a cohort study.

Interferon-γ release assays (IGRAs), which are widely used to diagnose tuberculosis (TB), cannot effectively discriminate latent TB infection (LTBI) from active TB (ATB). This study aimed to identify potential antigen-specific biomarkers for differentiating LTBI cases from ATB cases. Ongoing recruitment was conducted of individuals meeting study inclusion criteria at Beijing Chest Hospital from May 2020 to April 2022; 208 participants were enrolled and assigned to three groups: HC (60 healthy controls), LTBI (52 subjects with LTBI) and ATB (96 ATB patients). After participants were assigned to the discovery cohort (20 or 21 subjects/group), all others were assigned to the verification cohort. Discovery cohort blood levels of 40 chemokines were measured using Luminex assays to identify chemokines that could be used to discriminate LTBI cases from ATB cases; candidate biomarkers were verified using enzyme-linked immunosorbent assay-based testing of validation cohort samples. Luminex results revealed highest ATB group levels of numerous cytokines, growth factors and chemokines. Receiving operating characteristic curve-based analysis of 40 biomarkers revealed CCL8 (AUC = 0.890) and CXCL9 (AUC = 0.883) effectively discriminated between LTBI and TB cases; greatest diagnostic efficiency was obtained using both markers together (AUC = 0.929). Interpretation of CCL8 and CXCL9 levels for validation cohort IGRA-positive subjects (based on a 0.658-ng/ml cutoff) revealed ATB group CCL8-based sensitivity and specificity rates approaching 90.79% and 100.00%, respectively. TB-specific chemokines hold promise as ATB diagnostic biomarkers. Additional laboratory confirmation is needed to establish whether CCL8-based assays can differentiate between ATB and LTBI cases, especially for bacteriologically unconfirmed TB cases.

Enhancing the interferon-γ release assay through omission of nil and mitogen values

PurposeTo address the limited utility of the interferon (IFN)-γ release assay (IGRA) caused by its variability and inconsistency.MethodsThis retrospective cohort study was based on data obtained between 2011 and 2019. QuantiFERON-TB Gold-In-Tube was used to measure IFN-γ levels in nil, tuberculosis (TB) antigen, and mitogen tubes.ResultsOf 9,378 cases, 431 had active TB. The non-TB group comprised 1,513 IGRA-positive, 7,202 IGRA-negative, and 232 IGRA-indeterminate cases. Nil-tube IFN-γ levels were significantly higher in the active TB group (median = 0.18 IU/mL; interquartile range: 0.09–0.45 IU/mL) than in the IGRA-positive non-TB (0.11 IU/mL; 0.06–0.23 IU/mL) and IGRA-negative non-TB (0.09 IU/mL; 0.05–0.15 IU/mL) groups (P < 0.0001). From receiver operating characteristic analysis, TB antigen tube IFN-γ levels had higher diagnostic utility for active TB than TB antigen minus nil values. In a logistic regression analysis, active TB was the main driver of higher nil values. In the active TB group, after reclassifying the results based on a TB antigen tube IFN-γ level of 0.48 IU/mL, 14/36 cases with negative results and 15/19 cases with indeterminate results became positive, while 1/376 cases with positive results became negative. Overall, the sensitivity for detecting active TB improved from 87.2 to 93.7%.ConclusionThe results of our comprehensive assessment can aid in IGRA interpretation. Since nil values are governed by TB infection rather than reflecting background noise, TB antigen tube IFN-γ levels should be used without subtracting nil values. Despite indeterminate results, TB antigen tube IFN-γ levels can be informative.

Downregulation of monocyte miRNAs: implications for immune dysfunction and disease severity in drug-resistant tuberculosis.

Monocyte miRNAs govern both protective and pathological responses during tuberculosis (TB) through their differential expression and emerged as potent targets for biomarker discovery and host-directed therapeutics. Thus, this study examined the miRNA profile of sorted monocytes across the TB disease spectrum [drug-resistant TB (DR-TB), drug-sensitive TB (DS-TB), and latent TB] and in healthy individuals (HC) to understand the underlying pathophysiology and their regulatory mechanism. We sorted total monocytes including three subsets (HLA-DR+CD14+, HLA-DR+CD14+CD16+, and HLA-DR+CD16+cells) from peripheral blood mononuclear cells (PBMCs) of healthy and TB-infected individuals through flow cytometry and subjected them to NanoString-based miRNA profiling. The outcome was the differential expression of 107 miRNAs particularly the downregulation of miRNAs in the active TB groups (both drug-resistant and drug-sensitive). The miRNA profile revealed differential expression signatures: i) decline of miR-548m in DR-TB alone, ii) decline of miR-486-3p in active TB but significant elevation only in LTB iii) elevation of miR-132-3p only in active TB (DR-TB and DS-TB) and iv) elevation of miR-150-5p in DR-TB alone. The directionality of functions mediated by monocyte miRNAs from Gene Set Enrichment Analysis (GSEA) facilitated two phenomenal findings: i) a bidirectional response between active disease (activation profile in DR-TB and DS-TB compared to LTB and HC) and latent infection (suppression profile in LTB vs HC) and ii) hyper immune activation in the DR-TB group compared to DS-TB. Thus, monocyte miRNA signatures provide pathological clues for altered monocyte function, drug resistance, and disease severity. Further studies on monocyte miRNAs may shed light on the immune regulatory mechanism for tuberculosis.

Interleukin-4 expression is increased in patients with tuberculosis: A systematic review and meta-analysis.

Interleukin-4 (IL-4) is an important cytokine in the Th2 differentiation of CD4+ T cells, which modulates immune responses and participates in host defense against Mycobacterium tuberculosis. The present study aimed to evaluate the significance of IL-4 concentration in patients with tuberculosis. Data from this study will be helpful in understanding the immunological mechanisms of tuberculosis and in clinical practice. A data search was conducted from January 1995 to October 2022 in electronic bibliographic databases such as China National Knowledge Infrastructure, Wan Fang, Embase, Web of Science, and PubMed. The Newcastle-Ottawa Scale was used to assess the quality of the included studies. Heterogeneity between the studies was assessed using I2 statistics. Publication bias was determined by funnel plot, and Egger's test was used to confirm the presence of publication bias. All qualified studies and statistical analyses were performed using Stata 11.0. Fifty-one eligible studies comprising 4317 subjects were included in the meta-analysis. The results depicted a considerably increased level of serum IL-4 in patients with tuberculosis than in the controls (standard mean difference [SMD] = 0.630, [95% confidence interval (CI), 0.162-1.092]). However, there was no significant difference in plasma IL-4 levels between patients with TB and controls (SMD = 0.290, [95% CI, -0.430 to 1.010]). In addition, the infection status, TB focus location, drug resistance, race, research design type, and detection method divided the subjects into different subgroups for the meta-analysis. The results of the comparison of healthy controls and TB subjects showed that in the Asian population, the serum IL-4 level in patients with TB was higher than that in controls (SMD = 0.887, [95% CI, 0.202 to -1.573]) and patients with active TB as well as people with pulmonary TB showed increased serum IL-4 levels compared to controls (SMD = 0.689, [95% CI, 0.152-1.226]). In the case of the control group with latent TB, the active TB group had higher serum IL-4 levels than the control group (SMD = 0.920, [95% CI, 0.387-1.452]). The present meta-analysis showed that serum IL-4 varied in healthy individuals and patients with TB. Patients with active TB may also exhibit higher IL-4 concentrations.

2179. Multiple cytokine analysisbased on QuantiFERON-TB Gold Plus in different tuberculosis infection status:anexploratory study

Abstract Background To explore the differences of multiple cytokines secretion based on QuantiFERON-TB Gold Plus(QFT-Plus)antigens stimulation, and to screen cytokines with potential to differentiate tuberculosis infection status. Methods Twenty patients diagnosed microbiologically with active tuberculosis(ATB)in Peking Union Medical College Hospitaland Beijing Chest Hospital from February to April 2022 were enrolled.Fifteen healthcare workers(HCWs) with latent TB infection(LTBI),10 HCWs with previous TB and 10 HCWs as healthy control(HC) were recruited during the same period. After stimulated by QFT-Plus antigens,the secretion of interferon gamma(IFN-γ),tumor necrosisfactor-α(TNF-α), interleukin(IL)-2(IL-2), IL-6, IL-5, IL-10, interferon-inducible protein-10(IP-10), IL-1 receptor antagonist(IL-1Ra),chemokine(C-X-C)Ligand-1(CXCL-1)and macrophage chemoattractant protein 1(MCP-1)) in Plasma were measured by Luminex Assays. Results 1.After stimulation with QFT-Plus TB1 orTB2 antigens, the level of IFN-γin ATB and LTBI groups was significantly higher than that in HC group(p&amp;lt;0.05),while there was no significant difference between ATB and LTBI groups(p &amp;gt;0.05).Cytokines including IL-2,IL-1Ra, CXCL-1have the similar secretion patterns to IFN-γ. 2.After stimulation with TB1 antigen, the level of IP-10 in ATB group was significantly higher thanthat inLTBI,previous TBand HC groups(p&amp;lt;0.05).After stimulation with TB2 antigen, the level of IL-6in ATB group was significantly higher thanthat in LTBI and HC groups(p&amp;lt;0.05). 3.There was no significant difference incytokines secretion levels in QFT-Plus specific CD8 response(TB2-TB1) in most groups. Conclusion In addition to IFN-γ, thedetection of IL-2, IL-1Ra, CXCL-1by QFT-Plusmay havethe potential to detect tuberculosis infection.Combined detection of IP-10 and IL-6 levels may have the potential to differentiate ATB from LTBI Disclosures All Authors: No reported disclosures.

Aberrant expression of long non-coding RNAs in peripheral blood mononuclear cells response to tuberculosis in children.

We aimed to identify long non-coding RNAs (lncRNAs) aberrantly expressed in peripheral blood mononuclear cells (PBMCs) triggered by active tuberculosis (ATB), latent tuberculosis infection (LTBI), and healthy controls (HC). We examined lncRNAs expression in PBMCs isolated from children with ATB and LTBI, and from HC using RNA sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to explore the biological processes and signaling pathways of aberrantly expressed mRNAs. A total of 348 and 205 lncRNAs were differentially expressed in the ATB and LTBI groups, respectively, compared to the HC group. Compared to the LTBI group, 125 lncRNAs were differentially expressed in the ATB group. Compared to the HC group, 2317 mRNAs were differentially expressed in the ATB group, and 1093 mRNAs were differentially expressed in the LTBI group. Compared to the LTBI group, 2328 mRNAs were differentially expressed in the ATB group. The upregulated mRNAs were mainly enriched in neutrophil activation, neutrophil-mediated biological processes, and positive regulation of immune response in tuberculosis (TB), whereas the downregulated mRNAs were enriched in signaling pathways and structural processes, such as the Wnt signaling pathway and rDNA heterochromatin assembly. This is the first study on the differential expression of lncRNAs in PBMCs of children with TB. We identified significant differences in the expression profiles of lncRNAs and mRNAs in the PBMCs of children with ATB, LTBI, and HC, which has important implications for exploring lncRNAs as novel biomarkers for the diagnosis of TB. In addition, further experimental identification and validation of lncRNA roles could help elucidate the underlying mechanisms of Mycobacterium tuberculosis infection in children.

CD69 flow cytometry to complement interferon-γ release assay for active tuberculosis.

The interferon-γ (IFN-γ) release assay (IGRA) is widely used to diagnose tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb). However, indeterminate IGRA results due to "high Nil" or "low PHA" responses limit its clinical utility. We developed a novel assay using CD69 flow cytometry (FC) to complement IGRA. CD69 FC measures the surface CD69 expression on T cells prior to centrifugation to harvest the plasma for IGRA. T cell responses against Mtb antigen 1 (Ag1) or Ag2 were measured using three-color FC (CD3, CD4, and CD69) in TB (n=140) and non-TB groups (n=117). The cutoff values of Δ%CD69bright cells (stimulated minus unstimulated) for CD4+ and CD4- T cells were established based on healthy individuals (n=63). The assay performances of CD69 FC and IGRA were compared. In subjects with determinate IGRA results ("positive" or "negative"; n=216), the diagnostic accuracies of CD69 FC (90.3%) and IGRA (87.0%) were not significantly different (p=0.31). For indeterminate IGRA results (n=40), CD69 FC attained a diagnostic accuracy of 92.5%. The CD4+ /CD4- ratio within CD69bright T cells measured by CD69 FC was significantly higher (p < 0.05) in the active TB group (6.39 ± 132.05; n=72) than in other CD69 FC-positive subjects (2.84 ± 15.36; n=63) (p < 0.05), whereas CD8 responses expected by IGRA (difference of IFN-γ levels between Mtb Ag tubes) did not differ significantly (0.00 ± 9.18 and 0.00 ± 4.25, respectively, IU/ml; p=0.58). We demonstrated the potential of CD69 FC as a simple, rapid assay for clarifying indeterminate IGRA results and identifying active TB. With further improvements, CD69 FC may complement the IGRA to enhance TB risk stratification in the routine diagnostic workup.

Alterations in the nasopharyngeal microbiota associated with active and latent tuberculosis

ObjectiveTo investigate the characteristic of nasopharyngeal microbiota at different states of Mycobacterium tuberculosis (MTB) infection. MethodsParticipants were recruited from a chest hospital and were divided into three groups: the active tuberculosis (ATB) group, the latent TB infection (LTBI) group and the healthy control (HC) group. Nasopharyngeal microbiota was analyzed by 16S rRNA sequencing and clinical laboratory test results of ATB patients were collected and statistically analyzed. ResultsEleven ATB patients, 19 LTBI individuals and 18 healthy controls were included. Compared with LTBI group, Proteobacteria (P=0.04) and Gammaproteobacteria (P=0.01) increased in the ATB group. Compared with HC group, Pseudomonadales (P=0.03) and Moraxellaceae (P=0.04) increased, while Bacillales (P=0.04) and Lachnospiraceae (P=0.03) decreased in ATB group. Furthermore, Staphylococcus and Corynebacterium accounted for 70–80% in HC and LTBI groups. While in ATB group, they were less than 40%. Moreover, relative abundance of Corynebacterium, Corynebacteriaceae and Mycobacteriales was positively correlated with serum adenosine deaminase while negatively correlated with albumin, hemoglobin, and platelet counts in ATB patients. ConclusionsThe composition of nasopharyngeal microbiota changed significantly after MTB infection. The correlations between Corynebacterium and nutritional status (hemoglobin and albumin), immune-related molecules (adenosine deaminase) and inflammation-related indicators (platelet) in ATB patients deserve further exploration.

Analysis of real-time PCR Melanocortin 3 (MC3R) gene expression to identify new biomarkers inflammation in tuberculosis

BackgroundDiagnosis of tuberculosis (TB) in the era of technological sophistication requires accuracy and speed to provide as much information as possible so that TB treatment can be carried out quickly and precisely. New studies have also begun to be carried out to diagnose TB, one of which is by examining genes, either by looking at polymorphisms, mutations, or expressions. Several previous studies have confirmed the association of MC3R and TB genes with polymorphisms; MC3R is a gene that participates in the regulation of the inflammatory process and is also found in macrophages; therefore, we tried to analyze gene expression in the active TB group, household contacts, and healthy controls for looked at the differences between the three groups and confirmed the correlation of MC3R with TB by seeing which group's gene expression increased the most expression of the three groups so that the results can be considered as a TB diagnostic biomarker in the future.MethodsThis study included 122 people, 49 patients with confirmed TB, 46 close relatives of patients, and 27 healthy controls. This study used a real-time PCR technique to analyze MC3R gene expression in the three groups, and all data were analyzed using Bio-Rad CFXTM software version 3.1 and one-way ANOVA using SPSS 21.0.ResultsThe value of MC3R gene expression in the active TB group increased 3.6-fold in the healthy group (p = 0.143), and that of gene expression in the healthy control group increased 1.09-fold in the healthy group (p = 0.007).ConclusionThere is a relationship between MC3R and TB based on the results of gene expression analysis that increased in the active TB group compared to the household contact group and healthy controls.

Evaluating the diversity of circulating natural killer cells between active tuberculosis and latent tuberculosis infection

Tuberculosis (TB) is a leading global public health problem; however, the mechanisms underlying the immunopathology of TB progression are not well understood. It is currently believed that Mycobacterium tuberculosis (Mtb) infection can modify natural killer (NK) cell phenotypic signatures. Hence, our study was designed to investigate the diversity of circulating NK cells in patients with different TB infection status. NK subsets, as well as their expression of activating and inhibitory receptors between active TB (ATB) and latent TB infection (LTBI) were evaluated. There were significant differences in NK cell phenotypes between ATB, LTBI and healthy controls. Notably, the proportion of KLRG1 in NK cells (P = 0.036), as well as in their subsets CD56DimCD16+ (P = 0.046) and CD27+ (P = 0.027) NK cells, increased significantly in LTBI group than in ATB group; while Mtb specific IFN-γ+CD56BrightCD16Dim NK cells expressed higher KLRG1 in ATB than in LTBI (P = 0.027). However, the expression of activating receptor NKG2D in NK subsets showed no significant difference among the study groups. Our results suggest that different TB infection status are coupled with the diversity of NK cell compartments, and the expression of KLRG1 in NK cells may be a specific phenotype that modulates the progression of TB from latent to active.

QuantiFERON-TB Gold Plus Assay in Patients With Latent vs. Active Tuberculosis in a Low Incidence Setting: Level of IFN-γ, CD4/CD8 Responses, and Release of IL-2, IP-10, and MIG

ObjectivesWe analyzed the results of the QuantiFERON Glod Plus assay (QFT) and cytokine patterns associated with active tuberculosis (ATB) among patients with positive QFT.MethodsA total of 195 patients are QFT-positive, among which 24 had an ATB and 171 had a latent tuberculosis infection (LTBI). Interferon-gamma (IFN-γ) secretion was analyzed relative to interleukin-2 (IL-2), IFN-γ inducible protein or CXCL-10 (IP-10), and monokine induced by IFN-γ or CXCL-9 (MIG) secretion, and then compared between two sets of peptide antigens [tube 1 - cluster of differentiation 4 (CD4+) T cell stimulation; tube 2 - CD4+/CD8+ T cell response].ResultsHigher IFN-γ responses were measured in the ATB group (p = 0.0089). The results showed that there was a lower ratio of tube 1/tube 2 IFN-γ concentrations in the ATB group (p = 0.0009), and a median [interquartile ranges (IQR)] difference between the two sets at −0.82 IU/ml (−1.67 to 0.18) vs. −0.07 IU/ml (−0.035 to 0.11, p < 0.0001) in the ATB group compared to the LTBI group, respectively. In addition, patients with low ratios of IL-2/IFN-γ, IP-10/IFN-γ, and MIG/IFN-γ were much more likely to have ATB.ConclusionHigh levels of IFN-γ secretion, preferential IFN-γ response in tube 2, and lower secretion of IL-2, IP-10, and MIG release relative to IFN-γ secretion were more likely observed in subjects with ATB. These features of T cell response may be helpful in low prevalence settings to suspect ATB in patients tested positive for IFN-γ release assays (IGRA).

Impact of Vitamin D in Prophylaxis and Treatment in Tuberculosis Patients.

Vitamin D plays a crucial role in many infectious diseases, such as tuberculosis (TB), that remains one of the world’s top infectious killers with 1.5 million deaths from TB in 2021. Vitamin D suppresses the replication of Mycobacterium tuberculosis in vitro and showed a promising role in TB management as a result of its connection with oxidative balance. Our review encourages the possible in vivo benefit of a joint administration with other vitamins, such as vitamin A, which share a known antimycobacterial action with vitamin D. However, considering the low incidence of side effects even at high dosages and its low cost, it would be advisable to assess vitamin D level both in patients with active TB and high-risk groups and administer it, at least to reach sufficiency levels.

Prevalence and Risk Factors of Subclinical Tuberculosis in a Low-Incidence Setting in China.

Objectives: Subclinical tuberculosis (TB) represents a substantial proportion of individuals with TB disease, although limited evidence is available to understand the epidemiological characteristics of these cases. We aimed to explore the prevalence of subclinical patients with TB and identify the underlying association between the subclinical TB cases in the study setting and the Beijing genotype.Methods: A retrospective study was conducted among patients with incident TB at the Fifth People’s Hospital of Suzhou between January and December 2018. A total of 380 patients with TB were included in our analysis.Results: Of the 380 patients, 81.8% were active TB cases, whereas the other 18.2% were subclinical TB cases. Compared with patients aged 65 years and older, the risk of having subclinical TB is higher among younger patients. The use of smear, culture, and Xpert identified 3, 16, and 13 subclinical TB cases, respectively. When using a combination of positive culture and Xpert results, the sensitivity improved to 33.3%. In addition, the neutrophil-to-lymphocyte ratio was significantly elevated in the active TB group compared with that in the subclinical TB group. We also observed that the proportion of the Beijing genotype in the subclinical TB group was significantly lower than that in the active TB group.Conclusion: To conclude, our data demonstrate that approximately one-fifth of patients with TB were subclinical in Suzhou. Mycobacterium tuberculosis could be detected by the existing microbiologic diagnostics in one-third of patients with subclinical TB. The patients with subclinical TB are more prone to having low neutrophil-to-lymphocyte ratio values than those with active TB. Additionally, non-Beijing genotype strains are associated with subclinical TB.