Early Secretory Antigenic Target Research Articles

Integrating systemic immune-inflammation index, fibrinogen, and T-SPOT.TB for precision distinction of active pulmonary tuberculosis in the era of mycobacterial disease research.

The clinical challenge of differentiating suspected tuberculosis with positive T-SPOT.TB results persist. This study aims to investigate the utility of the Systemic Immune-Inflammation Index (SII), Fibrinogen, and T-SPOT.TB in distinguishing between active pulmonary tuberculosis (PTB) and non-tuberculous lung diseases. A retrospective analysis included 1,327 cases of active PTB with positive T-SPOT.TB results and 703 cases of non-tuberculous lung diseases from May 2016 to December 2020 at Meizhou People's Hospital. These were designated as the case group and the control group, respectively. The detection indicators of T-SPOT.TB: Early Secreted Antigenic Target 6 (ESAT-6), Culture Filtrate Protein 10 (CFP-10), as well as SII and Fibrinogen levels-were compared and analyzed for association and joint diagnostic value between the two groups. The case group showed higher values of ESAT-6, CFP-10, SII, and Fibrinogen compared to the control group (all p < 0.001). In the case group, SII and Fibrinogen did not correlate with ESAT-6 and CFP-10 (∣rs∣ all < 0.3) but were positively correlated with C-reactive protein (CRP; rs all > 0.3). SII and Fibrinogen values in smear-positive pulmonary tuberculosis were higher than in smear-negative cases (all p < 0.05). The optimal diagnostic thresholds for ESAT-6, CFP-10, SII, and Fibrinogen in differentiating between active PTB and non-tuberculous lung diseases were 21.50 SFCs/106 PBMC, 22.50 SFCs/106 PBMC, 2128.32, and 5.02 g/L, respectively. Regression logistic analysis showed that ESAT-6 < 21.5 (OR: 1.637, 95% CI: 1.311-2.043, p < 0.001), CFP-10 < 22.5 (OR: 3.918, 95% CI: 3.138-4.892, p = 0.025), SII < 2128.32 (OR: 0.763, 95% CI: 0.603-0.967, p < 0.001), and FIB < 5.02 (OR: 2.287, 95% CI: 1.865-2.806, p < 0.001) were independent risk factors for active PTB. The specificity for ESAT-6 + CFP-10, ESAT-6 + CFP-10 + SII, ESAT-6 + CFP-10 + FIB, and ESAT-6 + CFP-10 + SII + FIB was 82.5%, 83.2%, 95.8%, and 80.1%, respectively, while sensitivity was 52.6%, 53.0%, 55.8%, and 44.7%, and positive predictive values were 85.0%, 85.6%, 84.1%, and 89.6%, respectively. SII and Fibrinogen are positively correlated with the degree of tuberculosis inflammation and the bacterial load of Mycobacterium tuberculosis. The combined detection of SII, Fibrinogen, and T-SPOT.TB is significant in distinguishing between active PTB with positive T-SPOT.TB results and non-tuberculous lung diseases.

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

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. 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. 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. 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.

Surface-Activated Ti3C2Tx Adsorption of Acetylene Black Coupled with Polyaniline as a Signal Tag for the Detection of the ESAT-6 Antigen of Mycobacterium tuberculosis.

Early secretory antigenic target-6 (ESAT-6) is regarded as the most immunogenic protein produced by Mycobacterium tuberculosis, whose detection is of great clinical significance for tuberculosis diagnosis. However, the detection of the ESAT-6 antigen has been hampered by the expensive cost and complex experimental procedures, resulting in low sensitivity. Herein, we developed a titanium carbide (Ti3C2Tx)-based aptasensor for ESAT-6 detection utilizing a triple-signal amplification strategy. First, acetylene black (AB) was immobilized on Ti3C2Tx through a cross-linking reaction to form the Ti3C2Tx-AB-PAn nanocomposite. Meanwhile, AB served as a conductive bridge, and Ti3C2Tx can synergistically promote the electron transfer of PAn. Ti3C2Tx-AB-PAn exhibited outstanding conductivity, high electrochemical signals, and abundant sites for the loading of ESAT-6 binding aptamer II (EBA II) to form a novel signal tag. Second, N-CNTs were adsorbed on NiMn layered double hydride (NiMn LDH) nanoflowers to obtain NiMn LDH/N-CNTs, exhibiting excellent conductivity and preeminent stability to be used as electrode modification materials. Third, the biotinylated EBA (EBA I) was immobilized onto a streptavidin-coated sensing interface, forming an amplification platform for further signal enhancement. More importantly, as a result of the synergistic effect of the triple-signal amplification platform, the aptasensor exhibited a wide detection linear range from 10 fg mL-1 to 100 ng mL-1 and a detection limit of 4.07 fg mL-1 for ESAT-6. We envision that our aptasensor provides a way for the detection of ESAT-6 to assist in the diagnosis of tuberculosis.

Changes of Mycobacterium tuberculosis specific antigen-stimulated CD27−CD38+IFN-γ+CD4+ T cells before and after anti-tuberculosis treatment

BackgroundThe aim of the study was to investigate whether the expression of CD27−CD38+ in interferon (IFN)-γ+CD4+ T cells stimulated by the specific antigen early secreted antigenic target-6 (ESAT-6)/culture filter protein-10 (CFP-10) could be a potential new therapeutic evaluation indicator for anti-tuberculosis (TB) treatment.MethodsNewly diagnosed active pulmonary TB patients, latent TB infection (LTBI) and healthy controls were enrolled from January 2021 to December 2021. PTB patients were treated by standard anti-TB regimen 2HREZ/4HR (2 months of isoniazid (H), rifampin (R), ethambutol (E), and pyrazinamide (Z) followed by 4 months of isoniazid (H) and rifampin (R)). The difference of CD27−CD38+ expression in IFN-γ+CD4+ T cells before treatment, 2 months after treatment, and 6 months after treatment were compared.ResultsTotal 45 PTB patients, 38 LTBI cases and 43 healthy controls were enrolled. The expression of CD27−CD38+ decreased significantly after anti-TB treatment and was comparable with that in LTBI and healthy controls when the 6-month anti-TB treatment course was completed. The decline rate of CD27−CD38+ between 6 months after treatment and baseline was positively correlated with erythrocyte sedimentation rate (r = 0.766, P < 0.0001), C-reactive protein (r = 0.560, P = 0.003) and chest computerized tomography severity score (r = 0.632, P = 0.0005). The area under receiver operator characteristic curve of CD27−CD38+ in distinguish pulmonary TB patients before and after treatment was 0.779.ConclusionThe expression of CD27−CD38+ in ESAT-6/CFP-10 stimulated IFN-γ+CD4+T cells can well reflect the changes of the disease before and after anti-TB treatment, which is expected to be a potential new therapeutic evaluation index.Clinical Registry number chiCTR1800019966.

Microneedle patch-based enzyme-linked immunosorbent assay to quantify protein biomarkers of tuberculosis.

There is a clinical need for differential diagnosis of the latent versus active stages of tuberculosis (TB) disease by a simple-to-administer test. Alpha-crystallin (Acr) and early secretory antigenic target-6 (ESAT-6) are protein biomarkers associated with the latent and active stages of TB, respectively, and could be used for differential diagnosis. We therefore developed a microneedle patch (MNP) designed for application to the skin to quantify Acr and ESAT-6 in dermal interstitial fluid by enzyme-linked immunosorbent assay (ELISA). We fabricated mechanically strong microneedles made of polystyrene and coated them with capture antibodies against Acr and ESAT-6. We then optimized assay sensitivity to achieve a limit of detection of 750 pg/ml and 3,020 pg/ml for Acr and ESAT-6, respectively. This study demonstrates the feasibility of an MNP-based ELISA for differential diagnosis of latent TB disease.

Combined analysis of host IFN-γ, IL-2 and IP-10 as potential LTBI biomarkers in ESAT-6/CFP-10 stimulated blood

Background: Accurate diagnosis of latent tuberculosis infected (LTBI) individuals is important in identifying individuals at risk of developing active tuberculosis. Current diagnosis of LTBI routinely relies on the detection and measurement of immune responses using the Tuberculin Skin Test (TST) and interferon gamma release assays (IGRAs). However, IGRA, which detects Mycobacterium tuberculosis specific IFN-γ, is associated with frequent indeterminate results, particularly in immunosuppressed patients. There is a need to identify more sensitive LTBI point of care diagnostic biomarkers. The aim of this study was to assess the validity of early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) stimulated plasma to identify additional cytokines and chemokines as potential biomarkers of LTBI.Method: The levels of 27 cytokines and chemokines were measured by Bio-Plex Pro cytokine, chemokine and growth factor assay in ESAT-6 and CFP-10 co-stimulated plasma from 20 LTBI participants with positive IGRA (Quantiferon TB Gold plus) and 20 healthy controls with negative IGRA. Traditional ELISA was used to validate the abundance of the best performing markers in 70 LTBI and 72 healthy participants. All participants were HIV negative.Results: We found that Interleukin 1 receptor antagonist (IL1ra) (p = 0.0056), Interleukin 2 (IL-2) (p &amp;lt; 0.0001), Interleukin 13 (IL-13) (p &amp;lt; 0.0001), Interferon gamma-induced protein 10 (IP-10) (p &amp;lt; 0.0001), and Macrophage inflammatory protein-1 beta (MIP1b) (p = 0.0010) were significantly higher in stimulated plasma of LTBI compared to healthy individuals. Stimulated plasma IL-2 (cutoff 100 pg/mL), IP-10 (cutoff 300 pg/mL) and IL-13 (5 pg/mL) showed potential in diagnosing LTBI with PPV = 100%, 0.89.4%, and 80.9% and NPV = 86.9%, 0.85.7%, and 84.2%, respectively.Conclusion: Our data shows that co-stimulating whole blood with ESAT-6 and CFP-10 may help distinguish LTBI from healthy individuals. We also identified IL-2 and IP-10 as potential biomarkers that could be added to the currently used IFN-γ release assays in detection of LTBI.

A novel Mycobacterium Tuberculosis antigen, MTB48 enhances inflammatory response in LPS-induced RAW264.7 macrophage immune cells

Mtb (Mycobacterium tuberculosis) is a pathogenic bacterium that causes tuberculosis infection (TB). Mtb-secreted proteins have recently been investigated as virulence factors, as well as therapeutic and vaccine possibilities. The early-secreted antigen target MTB48 is one of these proteins that has been explored as a cocktail antigen in the serodiagnosis of active tuberculosis. However, there exists no information about the function or control of MTB48's inflammatory activity in macrophages at the site of inflammation. As a result, the goal of this research was to figure out what processes are involved in MTB48's function. MTB48 stimulated inflammation in LPS induced macrophages at both the protein and mRNA levels, which was interesting. MTB48 aided LPS induced IB phosphorylation and NF-κB translocation. MTB48 also led to the phosphorylation of MAPK signaling protein. These findings imply that MTB48 can enhance inflammatory activity via NF-κB and MAPK signaling by upregulating COX-2, iNOS, NO and PGE2. Many tuberculosis antigens have been tested for the development of rapid serological diagnosis. The results of this study suggest that MTB48 is a very high conservative antigen and is a major factor causing inflammatory reactions, suggesting that it can help control and diagnose tuberculosis.

Fluorescent nanodiamond immunosensors for clinical diagnostics of tuberculosis.

Fluorescent nanodiamonds (FNDs) are carbon nanoparticles containing a dense ensemble of nitrogen-vacancy defects as color centers. These centers have exceptional photostability and unique quantum properties, making them useful for ultrasensitive biosensing applications. This work employed FNDs conjugated with antibodies as magneto-optical immunosensors for tuberculosis (TB) diagnostics using competitive spin-enhanced lateral flow immunoassay (SELFIA). ESAT6 (6-kDa early secretory antigenic target) of Mycobacterium tuberculosis is a clinical marker of TB. We evaluated the assay's performance using the recombinant ESAT6 antigen and its antibodies noncovalently coated on FNDs. A detection limit of ∼0.02 ng mL-1 was achieved with the lateral flow membrane strip pre-structured with a narrow channel of 1 mm width. Adopting a cut-off value of 24.0 ng mm-1 for 100-nm FNDs on the strips, the method detected 49 out of 50 clinical samples with Mycobacterium tuberculosis complexes. In contrast, none of the assays for 10 clinical samples with non-tuberculous mycobacteria (NTM) isolates exhibited the presence of ESAT6. These results suggest that the SELFIA platform is applicable for TB detection and can differentiate TB from NTM infections, which also affect the human respiratory system. The FND-enabled immunosensing techniques are versatile and promising for early detection of TB and other diseases, opening a new avenue for biomedical applications of carbon-based nanomaterials.

HBHA-IGRA and cytotoxic mediators release assays for the diagnosis of cervical tuberculous lymphadenitis.

Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is a serious health problem in Tunisia. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis (CNTL). Here, we evaluated the performance of heparin-binding hemagglutinin (HBHA) interferon-gamma (IFN-γ) release assay (IGRA) for the diagnosis of CTL. In addition, we evaluated granzyme B, granulysin, and perforin release assays as CTL biomarkers and assessed their potential contribution to improve HBHA-IGRA performance. Peripheral blood mononuclear cells from CTL-suspected patients were stimulated with HBHA, early secreted antigenic target 6 (ESAT-6), or purified protein derivative (PPD) for 24 h in the presence of IL-7. Cytotoxic mediators and IFN-γ release were assessed by enzyme-linked immunosorbent assay. Receiver operating characteristic curves were used to evaluate the capacity of HBHA, ESAT-6, and PPD to discriminate between CTL (n = 27) and CNTL (n = 21). After applying bivariate and multivariate analyses, IFN-γ responses to HBHA appeared to offer the best distinction between CTL and CNTL, with an area under the curve of 0.9947, associated with 95.24% and 100% sensitivity and specificity, respectively. A principal component analysis showed clear clustering of the CTL versus the CNTL groups. This clustering was mainly attributed to HBHA-induced IFN-γ, PPD-induced granzyme B, and PPD-induced IFN-γ. These results thus suggest that the HBHA-IGRA provides high diagnostic accuracy for CTL versus CNTL, with high sensitivity and specificity. Combining HBHA-induced IFN-γ and PPD-induced granzyme B improves the accuracy to identify CTL.IMPORTANCECervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is currently a major health problem in Tunisia and in several regions around the world. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis. This study demonstrates the added value of the heparin-binding hemagglutinin-interferon-gamma release assay as an immunoassay in the context of CTL.

MXene-incorporated C60NPs and Au@Pt with dual-electric signal outputs for accurate detection of Mycobacterium tuberculosis ESAT-6 antigen

Rapid and effective detection of Mycobacterium tuberculosis (MTB) is the crux of minimizing tuberculosis (TB) spread. Consequently, a new electrochemical aptasensor based on dual-signal output for ultrasensitive detection of MTB early secreted antigenic target 6 (ESAT-6) antigen was developed. Especially, a new nanocomposite MXene/C60NPs/Au@Pt was synthesized for signal generation and amplification. In this biosensing architecture, dual independent signal outputs were achieved by coupling the electrochemical redox activity of fullerene nanoparticles (C60NPs) with the effective electrocatalytic activity of Au@Pt nanoparticles. MXene possesses a large specific surface area, allowing densely loaded of these two electroactive materials, further improved sensing capability. In addition, specific ESAT-6 antigen binding aptamers were attached to Au@Pt to create the tracer label. With a typical sandwich format along with the introduction of the gold nanoparticle-loaded molybdenum disulfide (MoS2–Au) as the sensing interface, the limit of detection (LOD) of the proposed aptasensor was 2.88 fg mL−1 (DPV measurement) and 13.50 fg mL−1 (IT measurement), respectively, with a broad linear range of 100 fg mL−1 to 50 ng mL−1. Significantly, it exhibited better specificity and accuracy with a sensitivity of 97.5% and a specificity of 96.7% to distinguish healthy donors, other lung diseases and TB patients compared to commercial ELISA assay, holding a promising prospect in clinical diagnosis.

Comparative Evaluation of Specific Antibody against Mycobacterium tuberculosis ESAT-6 Recombinant Antigen in Healthy Subject with Positive and Negative Skin Test.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). The laboratory diagnosis of the disease includes various bacteriologic and immunologic methods. Despite the effectiveness of many of these methods in diagnosing active TB, their high cost and time-consuming nature have led researchers to adopt more accurate and rapid screening methods based on specific antigens for M. tuberculosis. The present study aimed to measure specific antibody serum levels against the early secretory antigenic target 6 kDa (ESAT-6) recombinant protein in healthy people and compare it to TB patients. The target population included 27 TB patients and 87 healthy individuals with no clinical TB symptoms. The healthy population was divided into two groups, including positive purified protein derivative (PPD) and negative PPD (35 and 52 people, respectively), using the Tuberculin skin test. The specific antibody level against the ESAT-6 recombinant antigen and the PPD protein was measured using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) test. The results of the study showed that the majority of the healthy population with no symptoms of clinical TB and having negative skin test results did not have antibodies against the recombinant ESAT-6 (98%) and PPD (96%) antigens. On the other hand, there was a high level of the specific antibody of the ESAT-6 recombinant and PPD antigens in TB patients (77%). It is notable that in people with positive skin test results, the level of the antibody against the ESAT-6 recombinant antigen and PPD antigen was 94%. The results demonstrated that the ELISA method based on the measurement of antibodies against the ESAT-6 recombinant antigen can be a proper diagnostic method for rapid and accurate screening of healthy from infected people.

Validity of Examination of Complex Specific Antigen Mycobacterium Tuberculosis Rapid Immunochromatography Method in Patients with Pulmonary Tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis which can be transmitted from one person to another by inhaling droplets coughed or sneezed by an infected person. One of the most recent technologies today is the rapid immunochromatography (ICT) method for testing TB antigens. This test detects antigens secreted by Mycobacterium tuberculosis, namely the early secretory antigenic target 6 kDa protein (ESAT6), culture filtrate protein (CFP 10), and Mycobacterium tuberculosis protein (MPT64) encoded by region of difference genes (RDI, RD2, and RD3). The purpose of this study was to determine the validity of TB ICT antigens in diagnosing pulmonary tuberculosis. The research method used is a diagnostic test with a cross-sectional study design. The population of this study was all pulmonary TB patients who were examined at the Prof. Hospital Laboratory. W.Z. John. The samples used in this study amounted to 81 samples which were preceded by smear examination and then 47 samples of M. tuberculosis culture on Lowenstein Jensen media which was used as the gold standard, the results of the examination were 41 samples growing and 6 samples not growing. The sensitivity and specificity of the Rapid ICT TB antigen test were 75.61% and 100%, respectively. The conclusion of the rapid ICT TB antigen test has high validity, so it can be used as an alternative laboratory test for the diagnosis of pulmonary TB.

Analysis of a Combined HBHA and ESAT-6-Interferon-γ-Release Assay for the Diagnosis of Tuberculous Lymphadenopathies

The incidence of tuberculosis lymphadenopathy (TBLA) is increasing, and diagnostic procedures lack sensitivity and are often highly invasive. TBLA may be asymptomatic, and differential diagnosis with other adenopathies (ADPs) is difficult. We evaluated a blood-cell interferon-γ release assay (IGRA) with two different stage-specific mycobacterial antigens for the differential diagnosis of ADP suspected of mycobacterial origin. Twenty-one patients were included and divided into three groups: (1) cervical/axillar ADP (n = 8), (2) mediastinal ADP (n = 10), and (3) disseminated ADP (n = 3). The mycobacterial antigens used for the IGRA were the heparin-binding haemagglutinin (HBHA) and the early-secreted antigenic target-6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively. Diagnosis of TBLA based on microbiological results and/or response to anti-TB treatment was obtained for 15 patients. An IGRA profile highly suggestive of active TB (higher IFN-γ response to ESAT-6 compared to HBHA) was found for 3/6 TBLA patients from group 1, and for all the TBLA patients from groups 2 and 3, whereas this profile was not noticed in patients with a final alternative diagnosis. These results highlight the potential value of this combined HBHA/ESAT-6 IGRA as a triage test for the differential diagnosis of ADP.

Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10.

The current study reports on the development of a rapid and cost-effective TB-antigen diagnostic test for the detection of Mycobacterium biomarkers from non-sputum-based samples. Two gold nanoparticle (AuNP)-based rapid diagnostic tests (RDTs) in the form of lateral flow immunoassays (LFIAs) were developed for detection of immunodominant TB antigens, the 6 kDa early secreted antigen target EsxA (ESAT-6) and the 10 kDa culture filtrate protein EsxB (CFP-10). AuNPs were synthesized using the Turkevich method and characterized by UV-vis spectrophotometer and transmission electron microscope (TEM). The AuNP-detection probe conjugation conditions were determined by comparing the stability of 14 nm AuNPs at different pH conditions, following salt challenge. Thereafter, ESAT-6 and CFP-10 antibodies were conjugated to the AuNPs and used for the colorimetric detection of TB antigens. Selection of the best detection and capture antibody pairs was determined by Dot spotting. The limits of detection (LODs) for the LFIAs were evaluated by dry testing. TEM results showed that the 14 nm AuNPs were mostly spherical and well dispersed. The ESAT-6 LFIA prototype had an LOD of 0.0625 ng/mL versus the CFP-10 with an LOD of 7.69 ng/mL. Compared to other studies in the literature, the LOD was either similar or lower, outperforming them. Moreover, in some of the previous studies, an enrichment/extraction step was required to improve on the LOD. In this study, the LFIAs produced results within 15 min and could be suitable for use at PoCs either in clinics, mobile clinics, hospitals or at home by the end user. However, further studies need to be conducted to validate their use in clinical samples.

Convolutional neural network based on T-SPOT.TB assay promoting the discrimination between active tuberculosis and latent tuberculosis infection

The study aims to investigate the potential of convolutional neural network (CNN) based on spot image of T-SPOT assay for distinguishing active tuberculosis (ATB) from latent tuberculosis infection (LTBI). CNN was applied to recognize and classify T-SPOT spot image. Logistic regression was used to establish prediction model based on CNN. Areas under the receiver operating characteristic curve (AUCs) of early secreted antigenic target 6 (ESAT-6) CNN, culture filtrate protein 10 (CFP-10) CNN, and phytohemagglutinin (PHA) CNN were more than 0.7 in differentiating ATB from LTBI, while the performance of these indicators was significantly better than that of spot number. Furthermore, prediction model based on the combination of CNNs yielded an AUC of 0.898. The model presented a sensitivity of 85.76% and a specificity of 90.23%. The current study identified CNN based on T-SPOT spot image with the potential to serve as a tool for TB diagnostics.

High Positivity Rate of Urinary Mycobacterium tuberculosis Antigens Cocktail (ESAT-6, CFP-10, and MPT-64) in Active Tuberculosis Patients With and Without Human Immunodeficiency Virus Infection: A Cross-Sectional Study.

Human immunodeficiency virus (HIV) infection is a risk factor for the occurrence of a large of Mycobacterium tuberculosis (Mtb) antigen load in the body. The antigens cocktail namely early secretory antigenic target protein 6-kDa (ESAT-6), Culture filtrate protein 10 kDa (CFP-10), and Mycobacterium tuberculosis protein 64 (MPT-64) are secreted by Mtb during replication, hence, their concentration increase in patients with active Tuberculosis (TB). This increased levels facilitates their entry into the systemic circulation, followed by secretion by the glomerulus into the urine. The aim of this study was to determine the positivity rate of the urinary Mtb antigens cocktail between TB patients with and without HIV infection. This is an observational descriptive comparative study conducted with a cross-sectional design. Random urine samples were collected from patients diagnosed with active TB in Dr. Hasan Sadikin Bandung Hospital in 2021. The subjects were divided into 2 groups, TB-HIV group and TB without HIV group. The samples were tested using the quantitative immunochromatography method. Sixty active TB patients consisting of TB patients with HIV infection (n = 30) and TB patients without HIV infection (n = 30). The positivity in the urinary Mtb antigens cocktail was 93.3% for TB-HIV group and 100% for TB without HIV group (P = .492). The median concentration of urinary Mtb antigens cocktail in TB patients without HIV infection was higher than that of TB patients with HIV infection (137.73 ng/mL vs 96.69 ng/mL, respectively; P = .001). There was no significant difference in the positivity rate, meanwhile, there was a significant difference in concentration of the urinary Mtb antigens cocktail between active TB patients with and without HIV infection. Interestingly, this urinary Mtb antigens cocktail can be found in both groups without being affected by the patient's immune condition, thus becoming a test to assist diagnose active TB.

Development of diagnostic algorithm using machine learning for distinguishing between active tuberculosis and latent tuberculosis infection

BackgroundThe discrimination between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging. The present study aims to investigate the value of diagnostic models established by machine learning based on multiple laboratory data for distinguishing Mycobacterium tuberculosis (Mtb) infection status.MethodsT-SPOT, lymphocyte characteristic detection, and routine laboratory tests were performed on participants. Diagnostic models were built according to various algorithms.ResultsA total of 892 participants (468 ATB and 424 LTBI) and another 263 participants (125 ATB and 138 LTBI), were respectively enrolled at Tongji Hospital (discovery cohort) and Sino-French New City Hospital (validation cohort). Receiver operating characteristic (ROC) curve analysis showed that the value of individual indicator for differentiating ATB from LTBI was limited (area under the ROC curve (AUC) < 0.8). A total of 28 models were successfully established using machine learning. Among them, the AUCs of 25 models were more than 0.9 in test set. It was found that conditional random forests (cforest) model, based on the implementation of the random forest and bagging ensemble algorithms utilizing conditional inference trees as base learners, presented best discriminative power in segregating ATB from LTBI. Specially, cforest model presented an AUC of 0.978, with the sensitivity of 93.39% and the specificity of 91.18%. Mtb-specific response represented by early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) spot-forming cell (SFC) in T-SPOT assay, as well as global adaptive immunity assessed by CD4 cell IFN-γ secretion, CD8 cell IFN-γ secretion, and CD4 cell number, were found to contribute greatly to the cforest model. Superior performance obtained in the discovery cohort was further confirmed in the validation cohort. The sensitivity and specificity of cforest model in validation set were 92.80% and 89.86%, respectively.ConclusionsCforest model developed upon machine learning could serve as a valuable and prospective tool for identifying Mtb infection status. The present study provided a novel and viable idea for realizing the clinical diagnostic application of the combination of machine learning and laboratory findings.

High accuracy of recombinant fusion protein early secretory antigenic target protein 6‐culture filtrate protein 10 skin test for the detection of tuberculosis infection: a phase III, multi‐centered, double‐blind, hospital‐based, randomized controlled trial

ObjectivesTo verify the diagnostic utility of recombinant fusion protein ESAT6-CPF10 (EC), a novel skin test reagent to detect Mycobacterium tuberculosis infection. MethodsA multi-centered, double-blind, randomized controlled trial was conducted from December 17, 2015, to March 2, 2018. Participants involved in this study included those with active tuberculosis (TB), suspected pulmonary TB, or non-TB pulmonary disease. Each participant received three tests simultaneously, TB-specific enzyme-linked immunospot assay (T-SPOT.TB), tuberculin skin test (TST), and EC skin test (ECST), and adverse events were reported. ResultsDiagnostic accuracy was analyzed using data from 1085 protocol-compliant participants. The sensitivities of the ECST, TST, and T-SPOT.TB were 91.2% (95% CI, 89.0-93.2%), 91.4% (95% CI, 89.1-93.3%), and 92.1% (95% CI, 89.9-93.9%), respectively. The specificities of the ECST (69.7%, 95% CI, 64.5-74.5%) and T-SPOT.TB (76.1%, 95% CI, 71.2-80.5%) were significantly higher than the TST (54.4%, 95% CI, 48.9-59.7%). The agreements between ECST and TST (kappa = 0.632) and between ECST and T-SPOT.TB (kappa = 0.780) were substantial. No severe adverse event was reported. ConclusionThe diagnostic performance of the ECST was close to the T-SPOT.TB assay in the detection of TB infection and indicated good potential for clinical application in common scenarios.

Antigen-Specific Intraocular Cytokine Responses Distinguish Ocular Tuberculosis From Undifferentiated Uveitis in Tuberculosis-Immunoreactive Patients

To compare antigen-specific intraocular immune responses between different clinical phenotypes of tuberculin skin test (TST)-positive and TST-negative uveitis. Single center, retrospective cross-sectional study. Patients requiring diagnostic or therapeutic vitrectomy for the management of intraocular inflammation were divided into 3 groups based on Standardization of Uveitis Nomenclature (SUN) classification criteria for tubercular uveitis. Group 1 included patients with ocular tuberculosis (OTB; n=23) who were TST-positive patients, met the SUN criteria, and/or had a polymerase chain reaction (PCR)-positive test for TB. Group 2 included patients with uveitis of unknown origin (UNK; n=24) who were undifferentiated TST-positive patients who had not met SUN criteria. Group 3 included non-TB uveitis patients (n=24) who were TST-negative either with or without a well-defined non-TB diagnosis. Total vitreous cells were activated with Mycobacterium tuberculosis-specific Early Secreted Antigenic Target-6 (ESAT-6) or the retinal autoantigen, interphotoreceptor retinoid-binding protein peptide (pIRBP 1-20), stained forintracellular interferon gamma (IFNγ), tumor necrosis factor-alfa (TNFα), and interleukin 17 (IL-17), and analyzed by flow cytometry. Antigen-specific single and dual (polyfunctional) cytokine responses to ESAT-6 and IRBP were compared between the 3 groups. All cytokine responses to ESAT-6 were higher in the UNK group compared with the non-TB control subjects, while all except IL-17 were comparable between the OTB and non-TB groups. Polyfunctional responses-IFNγ/IL-17 (P=.002), TNFα/IL-17 (P=.02), and TNFα/IFNγ (P=.01)-were significantly greater for UNK than the OTB group. Polyfunctional cells also produced more cytokine per cell than respective monofunctional cells. IRBP cytokine responses were comparable between different groups and were not affected by the clinical phenotype or duration of disease. The intraocular polyfunctional cytokine response is stronger in undifferentiated TST-positive uveitis than in OTB patients, likely representing an exaggerated anti-TB immune response rather than active infection.

Differential activation of innate and adaptive lymphocytes during latent or active infection with Mycobacterium tuberculosis.

Most individuals infected with Mycobacterium tuberculosis (Mtb) have latent tuberculosis (TB), which can be diagnosed with tests (such as the QuantiFERON-TB Gold test [QFT]) that detect the production of IFN-γ by memory T cells in response to the Mtb-specific antigens 6 kDa early secretory antigenic target EsxA (Rv3875) (ESAT-6), 10 kDa culture filtrate antigen EsxB (Rv3874) (CFP-10), and Mtb antigen of 7.7 kDa (Rv2654c) (TB7.7). However, the immunological mechanisms that determine if an individual will develop latent or active TB remain incompletely understood. Here we compared the response of innate and adaptive peripheral blood lymphocytes from healthy individuals without Mtb infection (QFT negative) and from individuals with latent (QFT positive) or active TB infection, to determine the characteristics of these cells that correlate with each condition. In active TB patients, the levels of IFN-γ that were produced in response to Mtb-specific antigens had high positive correlations with IL-1β, TNF-α, MCP-1, IL-6, IL-12p70, and IL-23, while the proinflammatory cytokines had high positive correlations between themselves and with IL-12p70 and IL-23. These correlations were not observed in QFT-negative or QFT-positive healthy volunteers. Activation with Mtb-soluble extract (a mixture of Mtb antigens and pathogen-associated molecular patterns [PAMPs]) increased the percentage of IFN-γ-/IL-17-producing NK cells and of IL-17-producing innate lymphoid cell 3 (ILC3) in the peripheral blood of active TB patients, but not of QFT-negative or QFT-positive healthy volunteers. Thus, active TB patients have both adaptive and innate lymphocyte subsets that produce characteristic cytokine profiles in response to Mtb-specific antigens or PAMPs. These profiles are not observed in uninfected individuals or in individuals with latent TB, suggesting that they are a response to active TB infection.