Evaluation of molecular diagnostic test for detection of adult pulmonary tuberculosis: A generic protocol.

BACKGROUND OBJECTIVES: Tuberculosis (TB) continues to be the second most-leading cause of death due to a single infectious agent as of 2022 after COVID-19. Many affordable new molecular diagnostic tools are being developed for early and more accurate diagnosis, especially for low-resource settings in low- and middle-income countries. In this context, there is a need to develop a standardized protocol for validation of new diagnostic tools. Here, we describe a generic protocol for multi-centric clinical evaluation of molecular diagnostic tests for adult pulmonary TB. METHODS: This protocol describes a cross-sectional study in TB reference laboratories in India. Adults (>18 yr) visitng the chest clinics or outpatient departments with symptoms of TB need to be enrolled consecutively till the required sample size of 150 culture positives and 470 culture negatives are met. Mycobacterium tuberculosis (Mtb) culture (mycobacteria growth indicator tube liquid culture) to be used under this protocol as the gold standard and Xpert MTB/RIF molecular test will be used as the comparator. The sputum samples will be tested by smear microscopy, Mtb culture, Xpert MTB/RIF and index molecular test as per the proposed algorithm. The specificity sensitivity, and positive/ negative predictive values are to be calculated for the index test with reference to the gold standard. DISCUSSION: TB diagnosis poses many challenges as it differs with type of disease, age group, clinical settings and type of diagnostic tests/kits used. Globally, different protocols are used by several investigators. This protocol provides standard methods for the validation of molecular tests for diagnosis of adult pulmonary TB, which can be adopted by investigators.

Vitamin D Status, VDR, and TLR Polymorphisms and Pulmonary Tuberculosis Epidemiology in Kazakhstan.

BACKGROUND: Tuberculosis (TB) and vitamin D deficiency remain major public health problems in Kazakhstan. Due to the high incidence of pulmonary tuberculosis in the country and based on the importance of vitamin D in the modulation of the immune response and the association of its deficiency with many health conditions, the aim of our research was to study the vitamin D status, VDR and TLR gene polymorphisms, and pulmonary tuberculosis epidemiology in Kazakhstan. METHODS: A case-control study included 411 individuals diagnosed with pulmonary TB and 686 controls with no family history of pulmonary tuberculosis. Concentrations of serum vitamin D (25-(OH)D) levels were measured by electrochemiluminescence immunoassay. The gene polymorphisms were determined by real-time polymerase chain reaction (PCR) allelic discrimination assay using TaqMan probes. The association between the risk of pulmonary TB and polymorphisms was evaluated using multimodal logistic regression and assessed with the ORs, corresponding to 95% Cis, and the significance level was determined as p < 0.05. RESULTS: 1097 individuals were recruited from 3 different regions of Kazakhstan. Biochemical data showed vitamin D deficiency (25-(OH)D < 20 ng/mL) was present in both groups, with the case group accounting for almost 95% and 43.7% in controls. Epidemiological data revealed that socioeconomic factors such as BMI < 25 kg/m2 (p < 0.001), employment (p < 0.001), diabetes (p < 0.001), and vitamin D deficiency (p < 0.001) were statistically different between case and control groups. Logistic regression analysis, adjusted by sex, age, BMI, residence, employment, smoking, alcohol consumption, and diabetes, showed that T/T polymorphism of the VDR gene (rs1544410, OR = 1.97, 95% CI: 1.04-3.72, p = 0.03) and A/A polymorphism of the TLR8 gene (rs3764880, OR = 2.44, 95% CI: 1.20-4.98, p = 0.01) were associated with a high risk of developing pulmonary tuberculosis. CONCLUSIONS: Vitamin D deficiency remains prevalent in our study cohort and is associated with TB progression. Socioeconomic determinants such as unemployment, BMI under 25 kg/m2, and diabetes are the main risk factors for the development of pulmonary TB in our study. A/A polymorphism of TLR8 (rs3764880) and T/T polymorphism (BsmI, rs1544410) of VDR genes may act as biomarkers for pulmonary tuberculosis in the Kazakh population.

Genetic Variations of Angiotensinogen, Angiotensin Converting Enzyme, and Angiotensin Type 1 Receptor with the Risk of Pulmonary Tuberculosis.

There is little data regarding the impact of renin-angiotensin system (RAS) gene polymorphisms on tuberculosis. The current study designed to survey the possible association between RAS polymorphisms and the risk of pulmonary tuberculosis (PTB) in a sample of the southeast Iranian population. This case-control study was done on 170 PTB patients and 170 healthy subjects. The AGT rs699 C>T, ACE rs4341 C>G and AT1R rs5186 C>A variants were genotyped using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and ACE rs4646994 (287bp I/D) variant by PCR method. Regarding AT1R rs5186 A>C polymorphism, the findings revealed that AC genotype and C allele significantly decreased the risk of PTB (OR=0.39, 95% CI=0.22-0.67, p=0.001, and OR=0.53, 95% CI=0.25-0.72, p=0.002, C vs. A, respectively). The TC genotype and C allele of AGT rs699 T>C significantly associated with decreased the risk of PTB (OR=0.45, 95% CI=0.28-0.74, p=0.002, TC vs. TT and OR=0.51, 95% CI=0.32-0.80, p=0.005, C vs. T, respectively). The ID genotype of ACE 287bp I/D significantly increased the risk of PTB (OR=1.88, 95% CI=1.12-3.17, p=0.017). Our finding did not support an association between ACE rs4341 C>G variant and the risk of PTB. In summary, the findings revealed an association between AT1R rs5186 A>C, AGT rs699 T>C and ACE 287bp I/D polymorphisms and the risk of PTB in a sample of the southeast Iranian population. Further investigation with higher sample sizes and diverse ethnicities are required to confirm our findings.

Associations of genetic variants within TYK2 with pulmonary tuberculosis among Chinese population.

BACKGROUND: Pulmonary tuberculosis (PTB) is a common infectious disease caused by mycobacterium tuberculosis (MTB) and the present study aims to explore the associations of genetic variants within tyrosine kinases 2 (TYK2) with PTB incidence. METHODS: A population-based case control study including 168 smear-positive PTB cases and 251 controls was conducted. Five single nucleotide polymorphisms (SNPs) including rs280520, rs91755, rs2304256, rs12720270, rs280519 located within TYK2 gene were selected and MassARRAY® MALDI-TOF system was employed for genotyping. SPSS 19.0 was adopted for statistical analysis, non-conditional logistic regression was conducted. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were computed to estimate their contributions to PTB incidence. RESULTS: In the overall study population, rs91755 TT and rs280519 AA genotypes were found to be associated with reduced PTB risk (OR = 0.34, 95% CI: 0.16-0.72; OR = 0.38, 95% CI: 0.18-0.79, respectively). After stratification for sex, we found that among the male population, rs91755TG/TT, rs12720270AG/GG and rs280519AG/AA genotypes were associated with reduced PTB risk (OR = 0.41, 95% CI: 0.21-0.80; OR = 0.44, 95% CI: 0.21-0.94; OR = 0.42, 95% CI: 0.21-0.82, respectively). After stratification for age, we found that among those aged <60 years, rs91755TT and rs280519AA genotype were associated with reduced PTB risk (OR = 0.29, 95% CI: 0.09-0.90; OR = 0.34, 95% CI: 0.11-1.08, respectively); while rs2304256AC/AA genotype was associated with increased PTB risk (OR = 2.68, 95% CI: 1.05-6.85). Haplotype analysis revealed that AGAAG and ATCGA (Combined with rs280520, rs91755, rs2304256, rs12720270 and rs280519) were associated with increased (OR = 1.54, 95% CI: 1.01-2.37) and decreased PTB risk (OR = 0.70, 95% CI: 0.52-0.94), respectively. CONCLUSIONS: The genetic variants located within TYK2 including rs91755, rs12720270 and rs280519 were found to be associated with modified PTB risk and the SNPs had potential to be the biomarkers to predict PTB incidence risk.

Correlation of polymorphism in Toll-Like Receptor (TLR1 and TLR2) genes with susceptibility of pulmonary tuberculosis in Doda region of India.

BACKGROUND: Pulmonary tuberculosis has emerged as one of the leading causes of deaths across the globe. The prevalence of Mycobacterium tuberculosis has also shown an increasing trend over the time which may be attributed to the increase in multidrug resistant strains and HIV epidemics. There are several factors like change in the gene structure and cellular activities of the host and the bacterium which may have changed the host response towards tuberculosis. Additionally, the recent reports have suggested that Toll-Like Receptors (TLRs) play an important role in the activation of immune responses against various pathogens. Therefore, this study has been designed to investigate the possible correlation of TLR gene polymorphism and prevalence of pulmonary tuberculosis. METHOD: This study investigates 300 samples collected from patients with pulmonary tuberculosis (150) and healthy controls (150) from the Doda region of Jammu, India. For analysis purpose, DNA from the collected samples were isolated and subjected to sequence specific PCR amplification of TLR-1 and TLR-2 genes. The amplicons of TLR-1 and TLR-2 were further digested with restriction enzymes PvuII and Xbal, respectively, and visualized on agarose gel, subsequently. RESULT: The results suggest that frequency of TLR2 gene polymorphism (73.9%) is high in the patients below the age of 50 years, whereas, frequency of TLR-1 gene polymorphism is high (71%) in the patients above 50 years of age (p = 0.005). Further, the restriction digestion analysis of TLR1 genes has shown that nearly 78% of the confirmed normal cases exhibit homozygous normal conditions followed by 12% cases with heterozygous conditions and 10% cases of homozygous mutants. Similarly for TLR2 genes, nearly 78.6% of the confirmed normal cases have shown homozygous normal conditions followed heterozygous conditions (12.6%) and homozygous mutants (8.6%). CONCLUSION: This study establishes a preliminary correlation between TLR polymorphism and tuberculosis.

In-vivo expressed mycobacterial transcripts as diagnostic targets for pulmonary tuberculosis.

The nucleic acid amplification tests (NAATs) such as Xpert MTB/RIF have transformed the TB diagnostic field by significantly increasing the case detection. However, newer improved diagnostic assays are still needed to meet the WHO targets to end TB. Present study is based on a novel approach of utilizing the in-vivo expressed specific mycobacterial transcriptomic biomarkers for the diagnosis of pulmonary tuberculosis (PTB). Total 61 subjects were recruited including smear positive (smear+; n = 15), smear negative (smear-; n = 30) PTB patients and disease controls (n = 16). Transcripts of three mycobacterial genes Rv0986, Rv0971c and Rv3121 were analyzed using real time PCR (qRT-PCR) in sputum samples. qRT-PCR with Rv0986, Rv0971c and Rv3121 identified smear + PTB patients with 100 %, 78.6 % and 86.7 % sensitivity respectively. In smear- PTB patients, both Rv0986 and Rv0971c based qRT-PCR resulted in 63 %, sensitivity whereas Rv3121 identified these patients with ∼40 % sensitivity only. The sensitivity of the assay for smear-patients increased to 85 % when combinatorial analysis of qRT-PCR data for all the three genes was used. Thus, in-vivo expressed mycobacterial transcripts have promising potential as biomarkers for PTB diagnosis.

SLCO1B1 Polymorphisms are Associated with the Susceptibility to Pulmonary Tuberculosis in Chinese Females.

This study aimed to evaluate the role of SLCO1B1 polymorphisms in pulmonary tuberculosis (PTB) risk among Chinese patients. This study comprised 600 PTB patients (mean age: 37.43 ± 12.73 years) and 600 healthy controls (mean age: 37.39 ± 12.57 years) from a Chinese population. The SLCO1B1 rs2306283 and rs4149056 polymorphisms were detected using TaqMan genotyping assay. Chi-square (χ2) test was applied to calculate the Hardy-Weinberg Equilibrium (HWE) among controls. Logistic regression analysis was used to examine the odds ratio (OR) and 95% confidence interval (CI). After adjustment for age and gender, the frequency of rs4149056-C was significantly higher in PTB group (P = 0.017, OR = 1.375, 95% CI 1.058-1.786); meanwhile, rs4149056 was associated with increased PTB risk in dominant model (P = 0.015, OR = 1.424, 95% CI 1.072-1.892). The frequency and genotype of rs2306283 showed no significant difference between the two groups. In stratified analysis, rs2306283-GG showed notable susceptibility to PTB (P = 0.027, OR = 1.563, 95% CI 1.051-2.323 in recessive model) in females; rs4149056-C was also significantly higher in female PTB group (P = 0.039, OR = 1.741, 95% CI 1.028-2.948). Neither of rs2306283 and rs4149056 polymorphisms was associated with PTB risk in males. A haplotype analysis showed that patients carrying at least one SLCO1B1*15 haplotype had higher PTB risk (P = 0.004, OR = 1.527, 95% CI 1.145-2.034). SLCO1B1 polymorphisms are associated with the susceptibility to pulmonary tuberculosis in Chinese females.

Transcriptional profiling of human peripheral blood mononuclear cells in household contacts of pulmonary tuberculosis patients provides insights into mechanisms of Mycobacterium tuberculosis control and elimination.

Household contacts (HHCs) of patients with active tuberculosis (ATB) are at higher risk of Mycobacterium tuberculosis (M. tuberculosis) infection. However, the immune factors responsible for different defense responses in HHCs are unknown. Hence, we aimed to evaluate transcriptome signatures in human peripheral blood mononuclear cells (PBMCs) of HHCs to aid risk stratification. We recruited 112 HHCs of ATB patients and followed them for 6 years. Among the HHCs, only 2 developed ATB, while the remaining HHCs were classified into three groups: (1) HHC-1 group (n = 23): HHCs with consistently positive T-SPOT.TB test, negative chest radiograph, and no clinical symptoms or evidence of ATB during the 6-year follow-up period; (2) HHC-2 group (n = 15): HHCs with an initial positive T-SPOT result that later became negative without evidence of ATB; (3) HHC-3 group (n = 14): HHCs with a consistently negative T-SPOT.TB test and no clinical or radiological evidence of ATB. HHC-2 and HHC-3 were combined as HHC-23 group for analysis. RNA sequencing (RNA-seq) in PBMCs, with and without purified protein derivative (PPD) stimulation, identified significant differences in gene signatures between HHC-1 and HHC-23. Gene ontology analysis revealed functions related to bacterial pathogens, leukocyte chemotaxis, and inflammatory and cytokine responses. Modules associated with clinical features in the HHC-23 group were linked to the IL-17 signaling pathway, ferroptosis, complement and coagulation cascades, and the TNF signaling pathway. Validation using real-time PCR confirmed key genes like ATG-7, CXCL-3, and TNFRSF1B associated with infection outcomes in HHCs. Our research enhances understanding of disease mechanisms in HHCs. HHCs with persistent latent tuberculosis infection (HHC-1) showed significantly different gene expression compared to HHCs with no M. tuberculosis infection (HHC-23). These findings can help identify HHCs at risk of developing ATB and guide targeted public health interventions.

BACH1 promotes tissue necrosis and Mycobacterium tuberculosis susceptibility.

Oxidative stress triggers ferroptosis, a form of cellular necrosis characterized by iron-dependent lipid peroxidation, and has been implicated in Mycobacterium tuberculosis (Mtb) pathogenesis. We investigated whether Bach1, a transcription factor that represses multiple antioxidant genes, regulates host resistance to Mtb. We found that BACH1 expression is associated clinically with active pulmonary tuberculosis. Bach1 deletion in Mtb-infected mice increased glutathione levels and Gpx4 expression that inhibit lipid peroxidation. Bach1-/- macrophages exhibited increased resistance to Mtb-induced cell death, while Mtb-infected Bach1-deficient mice displayed reduced bacterial loads, pulmonary necrosis and lipid peroxidation concurrent with increased survival. Single-cell RNA-seq analysis of lungs from Mtb-infected Bach1-/- mice revealed an enrichment of genes associated with ferroptosis suppression. Bach1 depletion in Mtb-infected B6.Sst1S mice that display human-like necrotic lung pathology also markedly reduced necrosis and increased host resistance. These findings identify Bach1 as a key regulator of cellular and tissue necrosis and host resistance in Mtb infection.

Pulmonary tuberculosis biomarker miR-215-5p inhibits autophagosome-lysosome fusion in macrophages.

The normal autophagy flux is beneficial for the rapid elimination of phagocytic pathogens by macrophages. However, Mycobacterium tuberculosis interferes with the autophagy flux of macrophages to weaken their immune function and evade immune surveillance. In this study, we found that miRNA-215-5p was downregulated in tuberculosis patients. A potential diagnostic model for tuberculosis was established by combining miRNA-215-5p with three others differentially expressed microRNAs (miRNA-145-5p, miRNA-486-5p and miRNA-628-3p). Furthermore, we discovered that the up-regulated miRNA-215-5p could inhibit the maturation of autophagy by preventing the fusion of autophagosomes with lysosomes in macrophages. The role of TB-specific miRNA-215-5p in inhibiting auto-lysosome formation provides evidence of its potential role in Host-directed therapy for tuberculosis.

High and Low Levels of ABCB1 Expression Are Associated with Two Distinct Gene Signatures in Lung Tissue of Pulmonary TB Patients with High Inflammation Activity.

P-glycoprotein (encoded by the ABCB1 gene) has a dual role in regulating inflammation and reducing chemotherapy efficacy in various diseases, but there are few studies focused on pulmonary TB patients. In this study, our objective was to identify a list of genes that correlate with high and low levels of ABCB1 gene expression in the lungs of pulmonary TB patients with different activity of chronic granulomatous inflammation. We compared gene expression in two groups of samples (with moderate and high activity of tuberculomas) to identify their characteristic gene signatures. Gene expression levels were determined using quantitative PCR in samples of perifocal area of granulomas, which were obtained from 65 patients after surgical intervention. Subsequently, two distinct gene signatures associated with high inflammation activity were identified. The first signature demonstrated increased expression of HIF1a, TGM2, IL6, SOCS3, and STAT3, which correlated with high ABCB1 expression. The second signature was characterized by high expression of TNFa and CD163 and low expression of ABCB1. These results provide insight into various inflammatory mechanisms and association with P-gp gene expression in lung tissue of pulmonary TB patients and will be useful in the development of a host-directed therapy approach to improving the effectiveness of anti-TB treatment.

Diagnostic performance of microRNA-29a in active pulmonary tuberculosis: a systematic review and meta-analysis.

BACKGROUND: In recent years, more and more studies have shown that microRNA-29a (miRNA-29a) can be used as a potential biomarker for active tuberculosis, but the results of these studies are not consistent. OBJECTIVE: To comprehensively evaluate the value of miRNA-29a in the diagnosis of active tuberculosis by meta-analysis. METHODS: The databases of CNKI, WanFang, PubMed, The Cochrane Library, Web of Science and EMBASE were searched for relevant studies. Studies were screened strictly according to inclusion and exclusion criteria. QUADAS-2 scale was used to evaluate the quality of the included studies. Data were extracted and analyzed by Meta-DiSc 1.4 and Stata 16.0 software. RESULTS: 13 articles were included, including a total of 1598 subjects, including 872 active tuberculosis patients and 726 controls. The combined sensitivity and specificity of miRNA-29a in the diagnosis of active tuberculosis were 78 % and 76 %, respectively, and the area under the overall summary receiver operating characteristic curve was 0.8564. CONCLUSION: miRNA-29a can be used as a biomarker for the diagnosis of active tuberculosis.

Association between a single nucleotide polymorphism of the ALOX5 gene and susceptibility to multisystem tuberculosis in a Chinese Han population.

BACKGROUND: Host genetic single nucleotide polymorphisms can exert an influence susceptibility to tuberculosis infection. Previous investigations have demonstrated an association between the polymorphism in the ALOX5 gene and a range of diseases, encompassing not only noninfectious conditions like asthma, acute myocardial infarction, and cerebral infarction but also infections caused by various pathogens. However, the relationship between ALOX5 gene polymorphism and susceptibility to tuberculosis has received limited research attention. The ALOX5 gene encodes arachidonic acid 5-lipoxygenase(5-LO), which serves as the initiating catalyst in the generation of the inflammatory mediator leukotriene. Leukotrienes, products derived from the 5-LO pathway, are potent proinflammatory lipid mediators that assume a pivotal role in tuberculosis infections.Consequently, ALOX5 gene variants may be intricately associated with the pathogenesis of tuberculosis. In instances where the host exhibits immunocompromisation, infection with Mycobacterium tuberculosis can impact multiple systems. The involvement of multiple systems significantly augments the complexity of treatment and escalates patient mortality rates. Regrettably, the underlying mechanisms driving multisystem tuberculosis pathogenesis remain enigmatic, with clinicians paying scant attention to this aspect. Although the protein encoded by the ALOX5 gene represents a pivotal enzyme that catalyzes the metabolism of arachidonic acid into LXA4, and thereby plays a significant role in the inflammatory response during tuberculosis infection, studies investigating ALOX5 gene polymorphism and its association with susceptibility to multisystem tuberculosis in the Chinese Han population are exceptionally scarce. Therefore, the primary objective of this study is to comprehensively examine the correlation between ALOX5 gene polymorphisms and susceptibility to tuberculosis within the Chinese Han population, with particular emphasis on multisystemic tuberculosis. METHODS: A case‒control study design was employed, encompassing 382 individuals with pulmonary tuberculosis and 367 individuals with multisystemic tuberculosis as the case groups, along with 577 healthy controls.Whole blood DNA was extracted from all patients and healthy controls. Subsequently, three tag polymorphisms (rs2029253, rs7896431, rs2115819) within the ALOX5 gene were selectively identified and genotyped. RESULTS: After adjusting for age and sex, the presence of allele A at rs2029253 exhibited a pronounced association with an elevated risk of TB susceptibility when compared to the tuberculosis group and healthy control group. (ORa: 2.174, 95% CI: 1.827-2.587; Pa<0.001, respectively). Notably, the rs2029253 AG genotype and AA genotype displayed a significantly increased susceptibility to tuberculosis (ORa: 2.236, 95% CI: 1.769-2.825; Pa <0.001 and ORa: 4.577, 95% CI: 2.950-7.100; Pa <0.001, respectively) compared to the GG genotype. Moreover, in the analysis utilizing genetic models, rs2029253 also exhibited a markedly heightened susceptibility to tuberculosis in additive models, dominant models, and recessive models (Pa <0.001). Conversely, no significant association was observed between rs7896431, rs2115819, and tuberculosis. In the subgroup analysis, when comparing the pulmonary tuberculosis group with the healthy control group, we observed no significant disparities in the distribution frequencies of alleles, genotypes, and gene models (additive model, dominant model, and recessive model) for the three tag SNPs, with P-values were >0.05 after adjusting for age and sex. Additionally, we noted that the presence of allele A at rs2029253 was linked to an increased susceptibility to tuberculosis in the multisystemic tuberculosis group relative to the healthy control group (ORa: 2.292, 95% CI: 1.870-2.810; Pa<0.001). Similarly, the rs2029253 AG genotype, AA genotype, and gene models, including the additive model, dominant model, and recessive model, demonstrated a significantly elevated risk of tuberculosis susceptibility. CONCLUSIONS: The polymorphism in the ALOX5 gene is associated with susceptibility to multisystemic tuberculosis in the Chinese Han population.

Integration of Metabolomics and Transcriptomics Reveals Major Metabolic Pathways and Potential Biomarkers Involved in Pulmonary Tuberculosis and Pulmonary Tuberculosis-Complicated Diabetes.

Pulmonary tuberculosis (PTB) and diabetes mellitus (DM) are common chronic diseases that threaten human health. Patients with DM are susceptible to PTB, an important factor that aggravates the complications of diabetes. However, the molecular regulatory mechanism underlying the susceptibility of patients with DM to PTB infection remains unknown. In this study, healthy subjects, patients with primary PTB, and patients with primary PTB complicated by DM were recruited according to inclusion and exclusion criteria. Peripheral whole blood was collected, and alteration profiles and potential molecular mechanisms were further analyzed using integrated bioinformatics analysis of metabolomics and transcriptomics. Transcriptional data revealed that lipocalin 2 (LCN2), defensin alpha 1 (DEFA1), peptidoglycan recognition protein 1 (PGLYRP1), and integrin subunit alpha 2b (ITGA2B) were significantly upregulated, while chloride intracellular channel 3 (CLIC3) was significantly downregulated in the group with PTB and DM (PTB_DM) in contrast to the healthy control (HC) group. Additionally, the interleukin 17 (IL-17), phosphatidylinositol 3-kinase (PI3K)-AKT, and peroxisome proliferator-activated receptor (PPAR) signaling pathways are important for PTB infection and regulation of PTB-complicated diabetes. Metabolomic data showed that glycerophospholipid metabolism, carbon metabolism, and fat digestion and absorption processes were enriched in the differential metabolic analysis. Finally, integrated analysis of both metabolomic and transcriptomic data indicated that the NOTCH1/JAK/STAT signaling pathway is important in PTB complicated by DM. In conclusion, PTB infection altered the transcriptional and metabolic profiles of patients with DM. Metabolomic and transcriptomic changes were highly correlated in PTB patients with DM. Peripheral metabolite levels may be used as biomarkers for PTB management in patients with DM. IMPORTANCE The comorbidity of diabetes mellitus (DM) significantly increases the risk of tuberculosis infection and adverse tuberculosis treatment outcomes. Most previous studies have focused on the relationship between the effect of blood glucose control and the outcome of antituberculosis treatment in pulmonary tuberculosis (PTB) with DM (PTB_DM); however, early prediction and the underlying molecular mechanism of susceptibility to PTB infection in patients with DM remain unclear. In this study, transcriptome sequencing and untargeted metabolomics were performed to elucidate the key molecules and signaling pathways involved in PTB infection and the susceptibility of patients with diabetes to PTB. Our findings contribute to the development of vital diagnostic biomarkers for PTB or PTB_DM and provide a comprehensive understanding of molecular regulation during disease progression.

Identification of immune biomarkers in recent active pulmonary tuberculosis.

Tuberculosis (TB) has remained an unsolved problem and a major public health issue, particularly in developing countries. Pakistan is one of the countries with the highest tuberculosis infection rates globally. However, methods or biomarkers to detect early signs of TB infection are limited. Here, we characterized the mRNA profiles of immune responses in unstimulated Peripheral blood mononuclear cells obtained from treatment naïve patients with early signs of active pulmonary tuberculosis without previous history of clinical TB. We identified a unique mRNA profile in active TB compared to uninfected controls, including cytokines such as IL-27, IL-15, IL-2RA, IL-24, and TGFß, transcription factors such as STAT1 and NFATC1 and immune markers/receptors such as TLR4, IRF1, CD80, CD28, and PTGDR2 from an overall 84 different transcripts analyzed. Among 12 significant differentially expressed transcripts, we identified five gene signatures which included three upregulated IL-27, STAT1, TLR4 and two downregulated IL-24 and CD80 that best discriminate between active pulmonary TB and uninfected controls with AUC ranging from 0.9 to 1. Our data identified a molecular immune signature associated with the early stages of active pulmonary tuberculosis and it could be further investigated as a potential biomarker of pulmonary TB.

Association of PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms with pulmonary tuberculosis susceptibility in a Chinese population.

BACKGROUND: Autophagy can inhibit the survival of intracellular microorganisms including Mycobacterium tuberculosis (Mtb), and the PI3K/AKT/mTOR pathway plays a crucial role. This study investigated the association between PI3K/AKT/mTOR pathway autophagy-related gene polymorphisms and pulmonary tuberculosis (PTB) susceptibility. METHODS: KEGG pathway and gene ontology (GO) databases were searched for genes belonging to the PI3K/AKT/mTOR and autophagy pathways. Thirty SNPs in nine genes were identified and tested for their associations with tuberculosis in 130 patients with PTB and 271 controls. We constructed genetic risk scores (GRSs) and divided the participants into 3 subgroups based on their GRSs:0-5, 6-10, and 11-16. RESULTS: This analysis revealed that the AKT1 (rs12432802), RPTOR (rs11654508, rs12602885, rs2090204, rs2589144, and rs2672897), and TSC2 (rs2074969) polymorphisms were significantly associated with PTB risk. A decreasing trend was observed (P trend 0.020), in which a lower GRS was associated with a higher risk of PTB ([6-10] vs. [0-5]: OR (95%CI) 0.590 (0.374-0.931); [11-16] vs. [0-5]: OR (95%CI) 0.381 (0.160-0.906)). CONCLUSIONS: Polymorphisms in AKT1, RPTOR, and TSC2 may influence susceptibility to PTB.

Single nucleotide polymorphism rs2234711 of interferon gamma receptor 1 is associated with pulmonary tuberculosis in the population of North India.

Interferon-gamma (IFN-γ) is a pro-inflammatory cytokine playing essential role in immunity against tuberculosis (TB). IFN-γ performs function by binding to its receptor complex, consisting of two polypeptide chains viz. IFN-γ receptor 1 (IFN-γR1) and IFN-γ receptor 2 (IFN-γR2). Structural and functional deficiencies in IFN-γR1 can make individual vulnerable to even weak mycobacterial infections. Studies from different populations of the world have reported the association of single nucleotide polymorphisms (SNPs) present in IFNGR1 gene with TB, however, there are no such studies from India. Thus, the present study was designed to analyse the association of rs2234711 (C/T), rs7749390 (C/T) and rs1327475 (C/T) SNPs of IFNGR1 with TB in the population of North India. For the present study, 263 TB patients (at zero day of anti-tuberculosis therapy) and 256 healthy controls (HCs) were recruited. The genotyping of selected SNPs was done by high-resolution melting (HRM) curve analysis. The mRNA and surface expression data of IFNGR1 was extracted from our previous study and was grouped according to the genotypes of studied SNPs. The genotype 'TT' and 'T' allele of SNP rs2234711 (C/T) were found to be associated with TB in studied population ['T' vs 'C': OR (CI) = 1.79 (1.39-2.29); p-value < 0.0001]. The haplotypes 'C-C-C' of rs2234711-rs7749390-rs1327475 confers protection, while haplotype 'T-C-C' is a risk factor for TB in studied population. It was also found that 'TT' genotype of rs2234711 in HCs is associated with lower surface expression of IFNGR1 (p-value = 0.0078). In conclusion, 'TT' genotype is associated with lower surface expression of IFNGR1 and is increasing the susceptibility to TB in North Indian population.

A multi-center, prospective cohort study of whole blood gene expression in the tuberculosis-diabetes interaction.

Diabetes mellitus (DM) increases tuberculosis (TB) severity. We compared blood gene expression in adults with pulmonary TB, with or without diabetes mellitus (DM) from sites in Brazil and India. RNA sequencing (RNAseq) performed at baseline and during TB treatment. Publicly available baseline RNAseq data from South Africa and Romania reported by the TANDEM Consortium were also analyzed. Across the sites, differentially expressed genes varied for each condition (DM, TB, and TBDM) and no pattern classified any one group across all sites. A concise signature of TB disease was identified but this was expressed equally in TB and TBDM. Pathway enrichment analysis failed to distinguish TB from TBDM, although there was a trend for greater neutrophil and innate immune pathway activation in TBDM participants. Pathways associated with insulin resistance, metabolic dysfunction, diabetic complications, and chromosomal instability were positively correlated with glycohemoglobin. The immune response to pulmonary TB as reflected by whole blood gene expression is substantially similar with or without comorbid DM. Gene expression pathways associated with the microvascular and macrovascular complications of DM are upregulated during TB, supporting a syndemic interaction between these coprevalent diseases.

RTP4, a Biomarker Associated with Diagnosing Pulmonary Tuberculosis and Pan-Cancer Analysis.

Background: Pulmonary tuberculosis (PTB) is a global epidemic of infectious disease; the purpose of our study was to explore new potential biomarkers for the diagnosis of pulmonary tuberculosis and to use the biomarkers for further pan-cancer analysis. Methods: Four microarray gene expression sets were downloaded from the GEO public databases and conducted for further analysis. Healthy control (HC) samples and samples of pulmonary tuberculosis (PTB) were calculated with enrichment scores in folate biosynthesis pathways. The scores acted as a new phenotype combined with clinical information (control or PTB) for subsequent analysis. Weight gene coexpression network analysis (WGCNA) was used to seek the modules mostly related to PTB and folate biosynthesis in training sets. Twenty-nine coexistence genes were screened by intersecting the genes in the green-yellow module of GSE28623 and the brown module of GSE83456. We used the protein-protein interaction network analysis to narrow the gene range to search for hub genes. Then, we downloaded the unified and standardized pan-cancer data set from the UCSC database for correlations between biomarkers and prognosis and tumor stage differences. Results: Eventually, RTP4 was selected as a biomarker. To verify the reliability of this biomarker, an area under the ROC (AUC) was calculated in gene sets (GSE28623, GSE83456, and GSE34608). Lastly, to explore the difference in RTP4 expression before and after antituberculosis treatment, the GSE31348 gene set was enrolled to compare the expressions in weeks 0 and 26. The results showed significant differences between these two time points (p < 0.001). RTP4 was significantly upregulated in the pulmonary tuberculosis group compared to the healthy control group in three gene sets and downregulated after antituberculosis therapy in one gene set. These results suggest that RTP4 can be used as a potential biomarker in diagnosing tuberculosis. The results of pan-cancer analysis showed that high expression of RTP4 in 4 tumor types was positively correlated with poor prognosis and high expression of RTP4 in 6 tumor types was negatively correlated with poor prognosis. We found significant differences in the expression of the RTP4 gene at different stages in 5 types of tumors. Conclusion: RTP4 might be a new potential biomarker for diagnosing pulmonary tuberculosis.