BACKGROUND AND OBJECTIVE

Nontuberculous mycobacteria (NTM) lung disease appears like tuberculosis infection but is resistant to primary anti-tuberculosis drugs. Hence, patients whose sputum sample tests positive for acid-fast bacilli (AFB) and bacterial culture for several times should be assessed for colonization or infection with NTM in a damaged lung secondary to TB. In such cases, though drug-resistant TB may be adequately treated, treatment may need to be directed towards the NTM as well. In NTM therapy, the duration and choice of treatment agent is based upon the specific organism and disease extent. This study used one-step multiplex PCR (mPCR) assay for rapid differentiation of solid cultures in Ogawa medium as Mycobacterium tuberculosis (MTB) and/or NTM.

A total of 80 stocked isolates obtained from the Lung Center of the Philippines from January to December 2018 were screened for NTM in terms of growth in Ogawa medium, acid fastness, and MPT64 TB antigen test result. These were from sputum specimens of multidrug-resistant tuberculosis (MDR-TB) patients. DNA was extracted from cultures (n=55) grown in Ogawa medium and one-step mPCR was performed to identify NTM to the species level.

Out of 80 samples screened, a total of 55 isolates were identified as NTM. One-step mPCR identified 12.73% (7/55) as M. abscessus, 34.55% (19/55) as M. massiliense, 1.82% (1/55) as M. kansasii, and 50.91% (28/55) were identified only up to genus Mycobacteria spp. Neither M. avium complex nor M. intracellulare was identified among the samples tested.

One-step mPCR was able to identify isolates as MTB or NTM coinciding with the initial screening using MPT64 TB antigen test. Multiplex PCR has given a more specific identificati on to the species level. The use of mPCR in identifying MTB and clinically significant NTM’s is suitable for the adequate treatment of mycobacterial infection.

Early secreted antigenic target of 6 kDa protein (ESAT-6) is the major virulence factor of Mycobacterium tuberculosis (MTB), which can resist the clearance of MTB in bodies by inhibiting macrophage phagocytosis and autophagy reaction, thus impeding the immune defense function of the body against MTB infection. In addition, ESAT-6-induced apoptosis of macrophage and massive necrosis of innate immune cells can foster MTB proliferation and colonization, leading to systemic MTB infection. Moreover, ESAT-6 hampers the protective immune response of Th1 cells, reducing the secretion of pro-inflammatory cytokines and contributing to immune dysfunction, thus accelerating the course of MTB infection. During the process, the high immunogenicity of ESAT-6 can be leveraged as a dominant antigen in the development of new TB vaccines, making it a promising candidate with broad prospects for further development.
Humans ; Mycobacterium tuberculosis ; Vaccines ; Cytokines ; Apoptosis ; Autophagy ; Sepsis

Humans ; Mycobacterium tuberculosis/genetics* ; Microspheres ; Antitubercular Agents/pharmacology* ; Mutation ; Microbial Sensitivity Tests ; Fluoroquinolones ; Drug Resistance, Bacterial/genetics* ; Tuberculosis, Multidrug-Resistant/microbiology*

Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.
Animals ; Mice ; Rabbits ; Mycobacterium tuberculosis/genetics* ; Tuberculosis ; Antigens, Bacterial ; Cytokines ; Immunity, Cellular

Objectives To develop a multi-stage and multi-epitope vaccine, which consists of epitopes from the early secretory and latency-associated antigens of Mycobacterium tuberculosis (MTB). Methods The B-cell, cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes of 12 proteins were predicted using an immunoinformatics. The epitopes with antigenicity, without cytotoxicity and sensitization, were further screened to construct the multi-epitope vaccine. Furthermore, the proposed vaccine underwent physicochemical properties analysis and secondary structure prediction as well as 3D structure modeling, refinement and validation. Then the refined model was docked with TLR4. Finally, an immune simulation of the vaccine was carried out. Results The proposed vaccine, which consists of 12 B-cell, 11 CTL and 12 HTL epitopes, had a flexible and stable globular conformation as well as a thermostable and hydrophilic structure. A stable interaction of the vaccine with TLR4 was confirmed by molecular docking. The efficiency of the candidate vaccine to trigger effective cellular and humoral immune responses was assessed by immune simulation. Conclusion A multi-stage multi-epitope MTB vaccine construction strategy based on immunoinformatics is proposed, which is expected to prevent both active and latent MTB infection.
Mycobacterium tuberculosis/metabolism* ; Molecular Docking Simulation ; Toll-Like Receptor 4 ; Epitopes, T-Lymphocyte/chemistry* ; Epitopes, B-Lymphocyte/chemistry* ; Vaccines, Subunit/chemistry* ; Computational Biology/methods*

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.
Siderophores/metabolism* ; Iron/metabolism* ; Mycobacterium tuberculosis/metabolism* ; Cryoelectron Microscopy ; Adenosine Triphosphate/metabolism* ; ATP-Binding Cassette Transporters

Tuberculosis (TB) is an infectious disease that poses a serious threat to human health. About a quarter of the world's population were infected with Mycobacterium tuberculosis in 2020, and the majority of them were latently infected. Approximately 5%-10% of the population with latent tuberculosis infection may progress to active TB disease. Identifying latent TB infection from active TB by biomarkers and screening people with latent TB infection at high risk of progression for preventive treatment by biomarkers that can reliably predict the progression is one of the most effective strategies to control TB. This article reviews the progress of research on transcriptional and immunological biomarkers for identifying TB infection and predicting the progression from latent infection to active TB, with the aim of providing new ideas for tuberculosis control.
Humans ; Latent Tuberculosis/diagnosis* ; Tuberculosis/diagnosis* ; Mycobacterium tuberculosis/genetics* ; Biomarkers

Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.
Humans ; Rifampin/therapeutic use* ; Antibiotics, Antitubercular/therapeutic use* ; Mycobacterium tuberculosis ; Ethambutol/pharmacology* ; Isoniazid/pharmacology* ; Paraffin Embedding ; Retrospective Studies ; Cross-Sectional Studies ; Drug Resistance, Bacterial ; Sensitivity and Specificity ; Tuberculosis, Multidrug-Resistant/drug therapy*

Objective: To explore the influencing factors of the adverse outcome of pulmonary tuberculosis (PTB) among adolescents in Hangzhou City between 2005 and 2020. Methods: A retrospective cohort study was used to collect the information of adolescent PTB patients with the onset of PTB occurring from January 1, 2005 to December 31 in 12 designated tuberculosis hospitals in Hangzhou, mainly including demographic, epidemiological, clinical manifestations, bacteriological characteristics and other data, through the China Management Information System for Infectious Disease Surveillance and Reporting and the follow-up survey. All patients were followed up and the end time was December 31, 2021. Multivariate Cox regression model was used to analyze the factors affecting the adverse outcome of these patients. Results: The mean age of 4 921 adolescent PTB patients was (18.9±3.6) years old, and the number of male and female patients were 3 074 and 1 847 respectively. The adverse outcome accounted for 14.7% (725) of all patients. Multivariate Cox regression model showed that eight risk factors, including management model from patients themselves or family members (HR=5.87, 95%CI: 4.55-7.64), molecular biology examination positive for PTB (HR=4.62, 95%CI: 2.98-7.19), the number of sputum smears-positive≥1 (HR=3.72, 95%CI: 2.87-4.83), non-standardized therapy regimens of PTB (HR=3.69, 95%CI: 2.95-4.64), history of retreated PTB (HR=2.22, 95%CI: 1.46-3.36), migrant adolescents (HR=1.89, 95%CI: 1.54-2.34), the number of chest X-ray scan (HR=1.83, 95%CI: 1.65-2.04), and severe PTB (HR=1.38, 95%CI: 1.02-2.05), were associated with the adverse outcome of adolescent PTB patients. Age (HR=0.94, 95%CI: 0.92-0.96), as the only protective factor, was associated with the adverse outcome of these patients. Conclusion: The management mode, molecular biological examination, chemotherapy program, history of tuberculosis, sputum smear examination, severity of tuberculosis, household residence, chest X-ray examination and age are associated with the adverse outcomes of adolescent PTB patients in Hangzhou.
Humans ; Male ; Adolescent ; Female ; Young Adult ; Adult ; Retrospective Studies ; Tuberculosis, Pulmonary/drug therapy* ; Risk Factors ; Proportional Hazards Models ; Sputum ; Mycobacterium tuberculosis

Humans ; Tuberculosis, Extrapulmonary ; Retrospective Studies ; Paraffin Embedding ; Tuberculosis/diagnosis* ; Mycobacterium tuberculosis/genetics* ; Formaldehyde ; Sensitivity and Specificity ; Sputum