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Diagnosis of TB at early stages of HIV infection may lead to timely intervention for improving patient outcome. Antibodies to Mycobacterium tuberculosis recombinant RpfB protein and two immunodominant peptides of Rpf B protein were evaluated in the sera of HIV  + TB + , HIV + and HIV - pulmonary TB patients by ELISA. Serum antibodies from 90 % and 65 % of HIV + TB + patients reacted to recombinant RpfB protein and synthetic peptide RpfP1 respectively. Overall, this study shows that resuscitation promoting factor B elicits humoral antibody response in HIV + TB + co-infected individuals and be proposed as a potential biomarker for diagnosis of HIV + TB + patients, however further longitudinal follow up studies are warranted., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Non-sputum-based biomarker assay is urgently required as per WHO's target product pipeline for diagnosis of tuberculosis. Therefore, the current study was designed to evaluate the utility of previously identified proteins, encoded by in vivo expressed mycobacterial transcripts in pulmonary tuberculosis, as diagnostic targets for a serodiagnostic assay. A total of 300 subjects were recruited including smear+, smear- pulmonary tuberculosis (PTB) patients, sarcoidosis patients, lung cancer patients and healthy controls. Proteins encoded by eight in vivo expressed transcripts selected from previous study including those encoded by two topmost expressed and six RD transcripts (Rv0986, Rv0971, Rv1965, Rv1971, Rv2351c, Rv2657c, Rv2674, Rv3121) were analyzed for B-cell epitopes by peptide arrays/bioinformatics. Enzyme-linked immunosorbent assay was used to evaluate the antibody response against the selected peptides in sera from PTB and controls. Overall 12 peptides were selected for serodiagnosis. All the peptides were initially screened for their antibody response. The peptide with highest sensitivity and specificity was further assessed for its serodiagnostic ability in all the study subjects. The mean absorbance values for antibody response to selected peptide were significantly higher (p<0.001) in PTB patients as compared to healthy controls; however, the sensitivity for diagnosis of PTB was 31% for smear+ and 20% for smear- PTB patients. Thus, the peptides encoded by in vivo expressed transcripts elicited a significant antibody response, but are not suitable candidates for serodiagnosis of PTB., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Objectives: Bone tuberculosis (TB) is the third most common types of extrapulmonary tuberculosis. It is critical to understand mycobacterial adaptive strategies within bone lesions to identify mycobacterial factors that may have role in disease pathogenesis., Methods: Whole genome microarray was used to characterize the in-vivo transcriptome of Mycobacterium tuberculosis (M.tb) within bone TB specimens. Mycobacterial virulent proteins were identified by bioinformatic software. An in vitro osteoblast cell line model was used to study the role of these proteins in bone TB pathogenesis., Results: 914 mycobacterial genes were significantly overexpressed and 1688 were repressed in bone TB specimens. Pathway analysis of differentially expressed genes demonstrated a non-replicative and hypometabolic state of M.tb, reinforcement of the mycobacterial cell wall and induction of DNA damage repair responses, suggesting possible survival strategies of M.tb within bone. Bioinformatics mining of microarray data led to identification of five virulence proteins. The genes encoding these proteins were also upregulated in the in vitro MC3T3 osteoblast cell line model of bone TB. Further, exposure of osteoblast cells to two of these virulence proteins (Rv1046c and Rv3663c) significantly inhibited osteoblast differentiation., Conclusion: M.tb alters its transcriptome to establish infection in bone by upregulating certain virulence genes which play a key role in disturbing bone homeostasis., Competing Interests: Declaration of Competing Interest No conflicts of interest were declared., (Copyright © 2022 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Introduction: Diabetes is a potent risk factor for the activation of latent tuberculosis and worsens the tuberculosis (TB) treatment outcome. The major reason for mortality and morbidity in diabetic patients is due to their increased susceptibility to TB. Thus, the study was conducted to understand the crosstalk between M. tuberculosis and its host upon latent tuberculosis infection and under hyperglycemic conditions or diabetes., Methods: An animal model was employed to study the relationship between latent tuberculosis and diabetes. BCG immunization was done in mice before infection with M. tuberculosis , and latency was confirmed by bacillary load, histopathological changes in the lungs and gene expression of hspX, tgs1, tgs3 and tgs5 . Diabetes was then induced by a single high dose of streptozotocin (150 mg/kg body weight). Host factors, like various cytokines and MMPs (Matrix metalloproteinases), which play an important role in the containment of mycobacterial infection were studied in vivo and in vitro ., Results: A murine model of latent TB was developed, which was confirmed by CFU counts (<10 4 in the lungs and spleen) and granuloma formation in lungs in the latent TB group. Also, the gene expression of hspX, tgs1 , and tgs5 was upregulated, and after diabetes induction, blood glucose levels were >200 mg/dl. An in vitro study employing a THP-1 macrophage model of latent and active tuberculosis under normal and high glucose conditions showed that dormant bacilli were better contained in the presence of 5.5 mM glucose concentration as compared with active bacilli. However, the killing and restriction efficiency of macrophages decreased, and CFU counts increased significantly with an increase in glucose concentration., Discussion: The decreased levels of MCP-1, decreased expression of mmp-9 , and increased expression of mmp-1 in the latent group at high glucose concentrations could explain the failure of granuloma formation at high glucose conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Verma, Kaur, Luthra, Singh, Aggarwal, Verma, Radotra, Bhadada and Sharma.)

The evidence of an association between diabetes and latent tuberculosis infection (LTBI) remains limited and inconsistent. Thus, the study aims to delineate the role of diabetes in activation of latent tuberculosis infection. Murine model of latent tuberculosis and diabetes was developed, bacillary load and gene expression of resuscitation promoting factors (rpfA-E) along with histopathological changes in the lungs and spleen were studied. Treatment for LTBI [Rifampicin (RIF) + Isoniazid (INH)] was also given to latently infected mice with or without diabetes for 4 weeks. Diabetes was found to activate latent tuberculosis as the colony forming unit (CFU) counts were observed to be > 10 4 in lungs and spleen. The gene expression of hspX was downregulated and that of rpfB and rpfD was observed to be upregulated in latently infected mice with diabetes compared to those without diabetes. However, no significant reduction in the CFU counts was observed after 4 weeks of treatment with RIF and INH. Diabetes helps in the progression of LTBI to active disease mainly through altered expression of resuscitation promoting factors rpfB and rpfD, which can serve as important targets to reduce the shared burden of tuberculosis and diabetes., (© 2021. The Author(s).)

Aim: Key to the successful management of paediatric pulmonary tuberculosis (PTB) lies in the early detection and proper treatment. We evaluated the performances of modern diagnostic tests: loop-mediated isothermal amplification (LAMP-IS6110), Xpert MTB/RIF (Cepheid) and mycobacteria growth indicator tube (BACTEC MGIT 960 culture) against a modified version of international consensus diagnostic definition (i.e. composite reference standard (CRS))., Methods: A cross-sectional analytical study was conducted in a tertiary care hospital in North India from July 2016 to December 2017 involving 100 children <14 years with suspected PTB. Respiratory specimens (sputum, gastric lavage and/or bronchoalveolar lavage) were collected and subjected to LAMP-IS6110, Xpert MTB/RIF and BACTEC MGIT 960 culture assay., Results: Fifty-five children had confirmed and probable TB according to the CRS (prevalence = 58.5%). The sensitivity of BACTEC MGIT 960 culture, Xpert MTB/RIF and LAMP-IS6110 assay was 14%, 9.1% and 10.91%, respectively, when compared against the predefined CRS. The specificity for all these tests was 100%. When compared with BACTEC MGIT 960 culture as the gold standard, the LAMP-IS6110 assay and Xpert MTB/RIF assay had the sensitivity of 85.71% (95% CI: 42.13-99.64%) and 71.43% (95% CI: 29.04-96.33%), respectively. The specificity of both assays was 100%., Conclusions: We noted that LAMP-IS6110 performed better than Xpert MTB/RIF (Cepheid) in terms of sensitivity when compared against BACTEC MGIT 960 culture as reference standard, though specificity of both the tests was comparable. The diagnostic performance of BACTEC MGIT 960 culture was better than LAMP-IS6110 and Xpert MTB/RIF in paediatric PTB, when compared against CRS., (© 2021 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Background: Disseminated Mycobacterium avium complex (MAC) and Mycobacterium tuberculosis infections have almost similar clinical presentations but require different therapeutic management. Materials & methods: A duplex PCR was designed based on the sequence variation between the genes encoding catalase-peroxidase (KatG) of M. avium complex and M. tuberculosis , so as to discriminate MAC, M. tuberculosis and mixed mycobacterial (MAC + M. tuberculosis ) infections in HIV patients. Results: An accurate, single-step differential diagnosis of disseminated mycobacterial infections in HIV patients was achieved with specific detection of a single band each for M. avium (120 bp) and M. tuberculosis  (90 bp) and two bands for the mixed (120 and 90 bp) infections. Conclusion: katG gene-based duplex PCR can facilitate quick differential diagnosis of disseminated MAC and M. tuberculosis infections in HIV patients.

Objective: To elucidate disease-specific host protein profile in vitreous fluid of patients with intraocular inflammation due to tubercular uveitis (TBU)., Methods: Vitreous samples from 13 patients with TBU (group A), 7 with non-TBU (group B) and 9 with no uveitis (group C) were analysed by shotgun proteomics using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were subjected to pathway analysis using WEB-based Gene SeT Analysis Toolkit software., Results: Compared to control groups (B + C combined), group A (TBU) displayed 32 (11 upregulated, 21 downregulated) DEPs, which revealed an upregulation of coagulation cascades, complement and classic pathways, and downregulation of metabolism of carbohydrates, gluconeogenesis, glucose metabolism and glycolysis/gluconeogenesis pathways. When compared to group B (non-TBU) alone, TBU displayed 58 DEPs (21 upregulated, 37 downregulated), with an upregulation of apoptosis, KRAS signaling, diabetes pathways, classic pathways, and downregulation of MTORC1 signaling, glycolysis/gluconeogenesis, and glucose metabolism., Conclusion: This differential protein profile provides novel insights into the molecular mechanisms of TBU and a baseline to explore vitreous biomarkers to differentiate TBU from non-TBU, warranting future studies to identify and validate them as a diagnostic tool in TBU. The enriched pathways generate interesting hypotheses and drive further research., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Studies on human intraocular tuberculosis (IOTB) are extremely challenging. For understanding the pathogenesis of IOTB, it is important to investigate the mycobacterial transcriptional changes in ocular environment., Methods: Mice were challenged intravenously with Mycobacterium tuberculosis H37Rv and at 45 days post-infection, experimental IOTB was confirmed based on bacteriological and molecular assays. M. tuberculosis transcriptome was analyzed in the infected eyes using microarray technology. The identified M. tuberculosis signature genes were further validated and investigated in human IOTB samples using real-time polymerase chain reaction., Results: Following intravenous challenge with M. tuberculosis, 45% (5/12) mice showed bacilli in the eyes with positivity for M. tuberculosis ribonucleic acid in 100% (12/12), thus confirming the paucibacillary nature of IOTB similar to human IOTB. M. tuberculosis transcriptome in these infected eyes showed significant upregulation of 12 M. tuberculosis genes and five of these transcripts (Rv0962c, Rv0984, Rv2612c, Rv0974c and Rv0971c) were also identified in human clinically confirmed cases of IOTB., Conclusions: Differentially expressed mycobacterial genes identified in an intravenously challenged paucibacillary mouse IOTB model and presence of these transcripts in human IOTB samples highlight the possible role of these genes for survival of M. tuberculosis in the ocular environment, thus contributing to pathogenesis of IOTB., (© FEMS 2019.)

The upsurge of drug resistant tuberculosis is major health threat globally. To counteract, antimicrobial peptides are being explored as possible alternatives. However, certain limitations of peptide-based drugs such as potential toxicity, high cost and relatively low stability need to be addressed to enhance their clinical applicability. Use of computer predicted short active motifs of AMPs along with nanotechnology could not only overcome the limitations of AMPs but also potentiate their antimicrobial activity. Therefore, present study was proposed to in silico identify short antimicrobial motif (Pep-H) of human neutrophil peptide-1 (HNP-1) and explore its antimycobacterial activity in free form and using nanoparticles-based delivery systems. Based on colony forming unit analysis, motif Pep-H led to killing of more than 90% M. tb in vitro at 10 μg/ml, whereas, similar activity against intracellularly growing M. tb was observed at 5 μg/ml only. Thereafter, chitosan (244 nm) and gold nanoparticles (20 nm) were prepared for Pep-H with both the formulations showing minimal effects on the viability of human monocyte derived macrophages (MDMs) and RBC integrity. The antimycobacterial activity of Pep-H against intracellular mycobacteria was enhanced in both the nanoformulations as evident by significant reduction in CFU (>90%) at 5-10 times lower concentrations than that observed for free Pep-H. Thus, Pep-H is an effective antimycobacterial motif of HNP-1 and its activity is further enhanced by chitosan and gold nanoformulations.

Diabetes affects the presentation of tuberculosis including delayed clearance of the bacteria from host cells, however, the molecular changes which help survival of phagocytosed mycobacterium in the diabetic host are still not clear. The effect of in vitro high glucose concentrations on the proteome of the phagocytosed mycobacterium isolated from the human monocytic THP1 cell line derived macrophages has been investigated in the present study. Concurrent tuberculosis and hyperglycemia conditions were mimicked by growing M. tuberculosis infected THP1 cells under high glucose conditions. Phagocytosed bacilli were isolated 5 days post infection. Proteomics analysis of the isolated bacilli was done by two-dimensional gel electrophoresis followed by mass spectrometry. A total of 224 ± 18 protein spots were obtained out of which 10 were found to be differentially expressed under high glucose concentrations in comparison to normal glucose concentration. Further, identity of all the ten proteins namely Tgs3, Rv0547c, AcrA1, EsxU, Rv2219, Mpa, Rv2308, ORN, LucA, and Rv1414 was elucidated by peptide mass finger printing using Matrix-assisted laser desorption and ionization-mass spectrometry (MALDI/MS) assisted with MASCOT software. Though Tgs3, Rv0547c, AcrA1 and Mpa proteins have been demonstrated to play a major role in lipid metabolism under nitric oxide stress conditions, the functional role of rest of the differentially expressed proteins remains to be elucidated. Under hyperglycemic conditions in the host cells, differential expression of these proteins might help in the better survival of mycobacteria and can further act as suitable targets to design novel drugs for more effective therapy for comorbid tuberculosis and diabetes., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Intraocular tuberculosis (IOTB), an extrapulmonary manifestation of tuberculosis of the eye, has unique and varied clinical presentations with poorly understood pathogenesis. As it is a significant cause of inflammation and visual morbidity, particularly in TB endemic countries, it is essential to study the pathogenesis of IOTB. Clinical and histopathologic studies suggest the presence of Mycobacterium tuberculosis in retinal pigment epithelium (RPE) cells. Methods: A human retinal pigment epithelium (ARPE-19) cell line was infected with a virulent strain of M. tuberculosis (H37Rv). Electron microscopy and colony forming units (CFU) assay were performed to monitor the M. tuberculosis adherence, invasion, and intracellular replication, whereas confocal microscopy was done to study its intracellular fate in the RPE cells. To understand the pathogenesis, the transcriptional profile of M. tuberculosis in ARPE-19 cells was studied by whole genome microarray. Three upregulated M. tuberculosis transcripts were also examined in human IOTB vitreous samples. Results: Scanning electron micrographs of the infected ARPE-19 cells indicated adherence of bacilli, which were further observed to be internalized as monitored by transmission electron microscopy. The CFU assay showed that 22.7 and 8.4% of the initial inoculum of bacilli adhered and invaded the ARPE-19 cells, respectively, with an increase in fold CFU from 1 dpi (0.84) to 5dpi (6.58). The intracellular bacilli were co-localized with lysosomal-associated membrane protein-1 (LAMP-1) and LAMP-2 in ARPE-19 cells. The transcriptome study of intracellular bacilli showed that most of the upregulated transcripts correspond to the genes encoding the proteins involved in the processes such as adherence (e.g., Rv1759c and Rv1026 ), invasion (e.g., Rv1971 and Rv0169 ), virulence (e.g., Rv2844 and Rv0775 ), and intracellular survival (e.g., Rv1884c and Rv2450c) as well as regulators of various metabolic pathways. Two of the upregulated transcripts ( Rv1971, Rv1230c ) were also present in the vitreous samples of the IOTB patients. Conclusions: M. tuberculosis is phagocytosed by RPE cells and utilizes these cells for intracellular multiplication with the involvement of late endosomal/lysosomal compartments and alters its transcriptional profile plausibly for its intracellular adaptation and survival. The findings of the present study could be important to understanding the molecular pathogenesis of IOTB with a potential role in the development of diagnostics and therapeutics for IOTB.

Mycobacterium tuberculosis survives within the infected host cells by escaping phagolysosomal fusion and adaptation to hosts' intracellular milieu. Recently, the role of alveolar epithelial cells (AECs) in the survival of intracellular M. tuberculosis has gained importance. It has been reported that M. tuberculosis causes cytotoxicity in AECs, a phenotype attributed to M. tuberculosis virulence that could be due to the differential gene/protein expression of bacilli in these cells. Thus, the present study focused on comparative proteomic analysis of intracellular mycobacteria within macrophages and epithelial cells. Intracellular mycobacteria from infected alveolar epithelial (A549) and macrophage (THP-1) cell lines were harvested, and two-dimensional polyacrylamide gel electrophoresis of mycobacterial proteins was performed. The protein spots of interest were picked and MALDI-TOF/MS was performed to identify proteins. Differential expression of 11 mycobacterial proteins was found in infected AECs, out of which six proteins were confirmed by MS/MS analysis. The identified proteins mainly belonged to functional categories of virulence, detoxification and adaptation, and are known to take part in metabolism and respiration, maintenance of virulence and survival during stress. These proteins were predicted to contain peptide motifs possessing cell-penetration ability. Expression of such proteins by intracellular mycobacteria could be essential for survival and persistence in AECs in its virulent state, thus contributing to disease pathogenesis.

The ineffectiveness of anti-tuberculous therapy against dormant and drug-resistant mycobacteria demands scrutiny of alternative candidates like antimicrobial peptides having different mechanisms of action. The present study was designed to explore the activity of human beta defensin-1 (HBD-1) and its in silico identified short motif Pep-B against active and dormant Mycobacterium tuberculosis (M. tb) H37Rv. Activity of HBD-1 and Pep-B was determined against actively growing M. tb in vitro, inside monocyte-derived macrophages (MDMs) and dormant bacilli in in vitro potassium deficiency and human peripheral blood mononuclear cell (PBMC) granuloma models using colony-forming unit enumeration. The minimum inhibitory concentrations (MIC) of HBD-1 and Pep-B were found to be 2 and 20 μg/ml, respectively. These peptides also inhibited intracellular mycobacterial growth at concentrations lower than in vitro MICs along with increased IFN-γ levels. Although at higher concentration, HBD-1 (× 2 MIC) and Pep-B (× 2 MIC) led to decrease in in vitro dormant mycobacterial load as compared to rifampicin (× 25 MIC) and isoniazid (× 16 MIC). Similarly, both peptides showed higher killing efficacy against dormant mycobacteria inside granuloma as compared to rifampicin. Thus, the present study indicates that HBD-1 and its motif are effective antimicrobial players against both actively growing and dormant mycobacteria.

The present study was conducted to understand the aetiological link between tuberculosis (TB) and sarcoidosis. Sera from smear-positive TB subjects (n = 24), smear-negative TB subjects (n = 24), sarcoidosis patients (n = 24) and healthy controls (n = 24) were collected and circulating immune complexes were isolated. Sandwich ELISA was performed for detecting four highly specific mycobacterial regions of difference (RD) proteins (early secretory antigenic target 6 [ESAT6], 10 KDa culture filtrate protein [CFP10], 21 KDa CFP [CFP21] and mycobacterial protein from species TB [MPT 64]). Sensitivity and specificity was calculated, and receiver operating characteristic plots were plotted. Non-parametric Mann-Whitney U-test was used to calculate statistical significance. Seventy per cent of sarcoidosis patients showed the presence of immune complexes of mycobacterial RD proteins similar to that observed in the sera of smear-negative TB patients as opposed to antibody-based detection assay based on these RD proteins. Thus, immunoassays based on specific mycobacterial RD proteins also need to be developed and validated carefully to differentiate TB and sarcoidosis, a close mimic of smear-negative tuberculosis.

Pulmonary tuberculosis, the disease caused by Mycobacterium tuberculosis, still retains a top rank among the deadliest communicable diseases. Sputum expectorated during the disease continues to be a primary diagnostic specimen and also serves as a reservoir of bacteria. The expression pattern of mycobacteria in sputum will lead to an insight into bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Thus, in the present study, a whole genome microarray of Mycobacterium tuberculosis was used to elucidate the transcriptional profile of mycobacteria in the sputum samples of smear positive pulmonary tuberculosis patients. Overall, the mycobacteria in sputum appeared to be in a low energy and low replicative state as compared to in vitro grown log phase M. tb with downregulation of genes involved in ATP synthesis, aerobic respiration and translational machinery. Simultaneously, downregulation was also seen in the genes involved in secretion machinery of mycobacteria along with the downregulation of genes involved in the synthesis of phthiocerol dimycocerosate and phenol glycolipids. In contrast, the majority of the genes which showed an upregulation in sputum mycobacteria were of unknown function. Further identification of these genes may provide new insights into the mycobacterial behavior during this phase of infection and may help in deciphering candidates for development of better diagnostic and drug candidates.

The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings.

During intracellular residence in macrophages, mycobacterial lipids, namely phosphatidylinositol mannosides (PIM) and lipoarabinomannans, are expelled in the lung milieu to interact with host cells. PIM include a group of essential lipid components of Mycobacterium tuberculosis (M. tb) cell wall. Given that PIM function as mycobacterial adhesins for binding to host cells, the present study explored the consequences of interaction of M. tb PIM with alveolar epithelial cells (AEC). A 24-h PIM exposure at a concentration of 10 μg/mL to AEC conferred cytolysis to AEC via induction of apoptosis, suggesting their potential to alter alveolar epithelium integrity. The results also reflected that type I like AEC are more sensitive to cytolysis than type II AEC. PIM-treated AEC exhibited significant production of reactive oxygen species (ROS) and an immunosuppressive cytokine transforming growth factor-β (TGF-β) in the culture supernatants. Although AEC displayed constitutive mRNA transcripts for toll-like receptors (TLR2 and 4) as well as chemokines (IL-8 and MCP-1), no significant change in their expression was observed upon PIM treatment. Collectively, these results offer insights into PIM potential as M. tb virulence factor that might promote mycobacterial dissemination by causing cytolysis of AEC via increased production of ROS and TGF-β., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host antimycobacterial immune response and/or attenuate T-cell suppressive and macrophage-deactivating cytokines may prove to be useful in the treatment of tuberculosis. ESAT6, 6-kDa early secreted antigenic target, is a potent protective antigen and is considered as major target for long-lived memory cells. In the present study the immunotherapeutic potential of ESAT-6 has been evaluated in mouse model of experimental tuberculosis. In the present study the ESAT-6 protein was cloned in Escherichia coli using pET23a(+) plasmid and purified by Ni(2+)-NTA chromatography. Further, the immunotherapeutic potential of the recombinant ESAT-6 (in terms of CFU enumeration in the target organs and histopathological analysis of lungs) was evaluated against experimental tuberculosis. The recombinant ESAT-6 with C-terminal histidine-tag and free N-terminus mimics the natural form of ESAT-6 has been successfully cloned and purified. The recombinant ESAT-6 protein adjuvanted with dimethyl dioctadecylammonium bromide (DDA) moderately reduced the bacterial load in the target organs of infected mice. Further, the formulation (ESAT-6-DDA) was able to act synergistically when given in combination with antituberculosis drugs. This recombinant ESAT-6 showed good immunotherapeutic potential against experimental tuberculosis and can be used as an adjunct to the conventional antituberculosis chemotherapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Our study retrospectively reviewed the presentation, healing response, and functional results of eight cases of unilateral knee tuberculosis in children. The patients were evaluated for pain, instability, deformity, range of motion, and limb length discrepancy. Seven cases presented radiologically with Kerri and Martini's stage 1 or 2 and one in stage 4. Epiphyseal involvement was present in five cases. At final follow-up, all the knees were painless and stable. A full range of motion was recovered in the early stages. Limb length discrepancy was seen in six cases. Radiologically, despite clinical healing, the lytic lesions persisted for longer. Joint space was not reduced in seven cases at the initial presentation which was maintained following treatment. In the early stages, multidrug chemotherapy alone is sufficient and joints should be mobilized early in order to prevent stiffness. The lytic epiphyseal and metaphyseal cavities may persist for several months even after successful treatment. Limb length discrepancy is a common complication of knee tuberculosis in children.

Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex-specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 10(7) times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Objective: Detecting latent tubercular infection (LTBI) in sarcoidosis has important treatment implications. Traditionally tuberculin skin test (TST) is relied upon for this purpose. However, sarcoidosis is known to produce tuberculin anergy, which is not affected by high prevalence of tuberculosis (TB) infection. Interferon gamma release assays (IGRAs) have a higher sensitivity and specificity for detecting Mycobacterium tuberculosis (MTB) infection than the conventional TST as they utilize antigens specific for MTB complex. However, there is limited data regarding the performance of these tests in sarcoidosis, particularly in a setting of high population prevalence of LTBI. Herein, we studied the utility of IGRAs in the diagnostic work up of patients with sarcoidosis., Patients and Methods: Prospectively enrolled, biopsy-confirmed, glucocorticoid naive cases of pulmonary sarcoidosis; pulmonary and extrapulmonary TB; and, healthy controls underwent TST using 0.1 mL (1 tuberculin unit) of purified protein derivative RT23, and IGRA using QuantiFERON-TB-Gold In Tube assay (QFT) in blood. For TST an induration > or =10 mm was taken as positive. QFT was performed and interpreted as per the manufacturer's instructions., Results: We studied 38 patients with sarcoidosis (22 men, 16 women; mean age 42.5 years), 30 patients of TB (18 pulmonary, 12 extrapulmonary) and 30 healthy controls. Patients with sarcoidosis were more likely to have a negative TST compared to healthy controls (89.5% vs. 60%, p = 0.004) or TB (89.5% vs. 23.3%, p < 0.001). However, QFT positivity was not significantly different in sarcoidosis compared to controls (34.2% vs. 50%, p = 0.19), but was higher in TB patients as compared to sarcoidosis (60% vs. 34.2%, p = 0.03)., Conclusions: There is anergy to tuberculin in sarcoidosis. However, the results of QFT are not similarly affected. QFT continues to remain positive in many patients with sarcoidosis, and thus may be more accurate to detect LTBI in these patients.

From the time sarcoidosis has been described, there has always been a viewpoint that the disease is in some way related to tuberculosis (TB). Sarcoidosis is a granulomatous disease, which is likely a result of continued presentation of a poorly degradable antigen. Mycobacterium tuberculosis has been a very strong contender for this antigen. Besides the molecular studies demonstrating mycobacterial deoxyribonucleic acid (DNA) in the sarcoid tissue, assessment of immune responses against mycobacterial antigens provides a useful tool to study the role of mycobacteria in the pathogenesis of sarcoidosis. We reviewed the studies focussing on T-cell and B-cell responses to tubercular antigens in patients with sarcoidosis. Pooled data from various studies does provide a suggestive, though not unequivocal evidence in favour of mycobacteria as a cause of sarcoidosis. These findings not only reinforce the possible pathogenic role of mycobacterial antigens in sarcoidosis, but at the same time also limit the clinical utility of molecular and serological studies based on mycobacterial antigens in the differential diagnosis of TB from sarcoidosis, particularly in a country with high endemicity for TB.

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity., (2010 Elsevier Inc. All rights reserved.)

Tuberculin skin test (TST), an age old method is based on measuring delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). However, inspite of simplicity, ease and cost effectiveness, the usefulness of PPD test is limited due to its inability to distinguish among a protective immune response, latent infection and active tuberculosis disease. On the other hand, a skin test based on RD antigens would add advantages of a high specificity of antigens with the logistics of a skin test. However, except few reports, in vivo data of intradermal use of RD antigens for skin testing is limited. Therefore, in the present study, four M. tuberculosis (Mtb) specific antigens (ESAT6, CFP10, CFP21 and MPT64) were evaluated for their diagnostic utility based on DTH response. These antigens alone and their multiple combinations induced strong DTH response in Mtb infected guinea pigs and the response was negligible in BCG vaccinated and sham immunized animals.

ESAT6 has recently been demonstrated to cause haemolysis and macrophage lysis. Our studies demonstrate that ESAT6 causes cytolysis of type 1 and type 2 pneumocytes. Both types of pneumocytes express membrane laminin, and ESAT6 exhibits dose-dependent binding to both cell types and to purified human laminin. While minimal ESAT6 was detected on the surface of Mycobacterium tuberculosis grown in vitro, exogenously provided ESAT6 specifically associated with the bacterial cell surface, and the bacterium-associated ESAT6 retained its cytolytic ability. esat6 transcripts were upregulated approximately 4- to approximately 13-fold in bacteria replicating in type 1 cells, and approximately 3- to approximately 5 fold in type 2 cells. In vivo, laminin is primarily concentrated at the basolateral surface of pneumocytes where they rest on the basement membrane, which is composed primarily of laminin and collagen. The upregulation of esat6 transcripts in bacteria replicating in pneumocytes, the specific association of ESAT6 with the bacterial surface, the binding of ESAT6 to laminin and the lysis of pneumocytes by free and bacterium-associated ESAT6 together suggest a scenario wherein Mycobacterium tuberculosis replicating in pneumocytes may utilize surface ESAT6 to anchor onto the basolateral laminin-expressing surface of the pneumocytes, and damage the cells and the basement membrane to directly disseminate through the alveolar wall.

This study was designed to evaluate the immunotherapeutic potential of Mycobacterium tuberculosis Ag85AB emulsified with unmethylated CpG motif-containing oligonucleotide (CpG-ODN) and dimethyldioctadecylammonium bromide (DDA) adjuvants (Ag85AB-CpG-DDA) in conjunction with antituberculous drugs. Ag85 complex proteins of M. tuberculosis purified from total culture filtrate and purified proteins were emulsified with CpG-ODN and DDA adjuvants. Mice were infected with M. tuberculosis H37 Rv and left for 30 days to establish infection. These mice were named 'tuberculous mice'. Tuberculous mice were treated with Ag85AB-CpG-DDA alone or in conjunction with antituberculous drugs. Treatment of tuberculous mice with Ag85AB-CpG-DDA in conjunction with antituberculous drugs reduced significant bacilli burden in lung and spleen. Moreover, treatment of tuberculous mice with Ag85AB-CpG-DDA induced higher production of type-I cytokines, generated more CD44-positive T cells and suppresses secretion of IL-4 as compared with untreated animals. In conclusion, this study shows that Ag85AB-CpG-DDA formulation may act as a potential future therapeutic regimen in conjunction with antituberculous drugs.

Different combinations of ESAT-6, CFP-10, CFP-21, MPT-64, encoded by RD1 and RD2 of Mycobacterium tuberculosis were evaluated on the basis of antigenicity in PPD positive TB contacts and immunogenicity in C57BL/6J mice immunized with the combination of all four RD antigens. The peripheral blood mononuclear cells of TB contacts showed maximum recognition in response to the combination of ESAT-6+MPT-64 in terms of predominant lymphoproliferation, IFN-gamma levels and the number of responders. On the contrary, the combination of ESAT-6+CFP-21+MPT-64 was found to be most immunogenic based on both T-cell and antibody responses in immunized mice. Prophylactic potential of the selected combinations was assessed as supplementation vaccines to BCG against intravenous challenge with M. tuberculosis in mice. BCG supplementation with the selected combinations resulted in significantly greater protection as compared to BCG alone against experimental tuberculosis and thus appears to be a promising approach to enhance the protective efficacy of the existing vaccine.

The attempts to find an effective antituberculous subunit vaccine are based on the assumption that it must drive a Th1 response. In the absence of effective correlates of protection, a vast array of mycobacterial components are being evaluated worldwide either on the basis of their ability to be recognized by T lymphocytes in in vitro assays during early stage of animal or human infection (antigenicity) or their capacity to induce T cell response following immunization in animal models (immunogenicity). The putative vaccine candidates selected using either of these strategies are then subjected to challenge studies in different animal models to evaluate the protective efficacy. Here we review the outcome of this current scheme of selection of vaccine candidates using an 'antigenicity' or 'immunogenicity' criterion on the actual protective efficacy observed in experimental animal models. The possible implications for the success of some of the leading vaccine candidates in clinical trials will also be discussed.

The differences in specificity of human lung and peripheral lymphocytes for mycobacterial antigens (Ag) need to be evaluated in order to identify vaccine candidates against pulmonary tuberculosis (TB). Therefore, the present study examined the response to low molecular weight secretory proteins of Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) from minimal pulmonary TB and non-TB patients. Ag85A, Ag85B, culture filtrate protein (CFP)-31, CFP-22.5, CFP-21, M. tuberculosis protein-64 and an as yet uncharacterised 19 kDa protein were found to be predominantly recognised by BAL cells of TB patients on the basis of lymphocyte proliferation and significant interferon-gamma release. However, recognition of CFP-8, 6-kDa early secreted antigenic target, CFP-10, CFP-14.5, M. tuberculosis secretory protein-17 and five other as yet uncharacterised low molecular weight polypeptides was found to be high on the basis of lymphocyte proliferation at the level of PBMCs. Furthermore, BAL macrophages, and not blood monocytes, were found to produce nitric oxide (NO) in response to mycobacterial Ags. Among polypeptides predominantly recognised by BAL lymphocytes, only Ag85A and Ag85B were found to induce both NO and interleukin-12 (p40) by alveolar macrophages. In conclusion, the present results indicate heterogeneity in antigen recognition by bronchoalveolar lavage cells and peripheral mononuclear blood cells of minimal tuberculosis patients, and also suggest the utility of antigen 85 complex polypeptides for the development of a future mucosal antituberculous vaccine.

Secreted proteins encoded by different regions of difference (RDs) from the genome of Mycobacterium tuberculosis have been considered as attractive candidates for vaccination against tuberculosis owing to their absence in most BCG strains. In this study, the structural gene for the RD2 locus encoding protein CFP21 was PCR amplified and expressed as a fusion protein with hexahistidine residues in Escherichia coli. Expression of CFP21 in E. coli under transcriptional regulation of the T7 promoter yielded a protein located within inclusion bodies. The inclusion bodies were solubilized in the presence of 8M urea and the protein was purified to homogeneity under denaturing conditions at low pH using nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradient dialysis against a decreasing concentration of urea. The purified protein was shown to have esterase activity. CFP21 protein was evaluated for immunogenicity in C57BL/6J mice. We observed an elevated T cell proliferative response and production of IFN-gamma and IL-12 (p40). CFP21 also induced an optimum level of cytotoxic T cell activity and induced a strong humoral response as indicated by higher levels of specific IgG1 and IgG2a antibody isotypes. In addition, a moderate level of protection was observed against experimental tuberculosis. This is the first report describing esterase activity of the M. tuberculosis complex-restricted protein CFP21 and its protective potential against experimental tuberculosis.

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.

This study examined the role of intranasal vaccination with Mycobacterium tuberculosis antigen85 complex proteins formulated in dimethyldioctadecylammonium bromide against airway Mycobacterium tuberculosis challenge in mice. Intranasal vaccination with antigen85A and antigen85B induced a significantly higher level of interferon-gamma, interleukin-12 and interleukin-4 in cervical lymph nodes together with IgA and IgG, predominantly IgG2a isotype in nasal secretion over subcutaneous vaccination. Further, intranasal vaccination with antigen85A and antigen85B imparted protection comparable with that obtained from intranasal or subcutaneous Mycobacterium bovis bacillus Calmette-Guerin immunization. These results suggest that mucosal vaccination via the intranasal route is of importance in the development of vaccine for tuberculosis.

The cytotoxic T-lymphocyte (CTL) responses to culture filtrate antigens of Mycobacterium tuberculosis H(37)Rv (RvCFP) and purified protein derivative (PPD) were investigated in active pulmonary tuberculosis patients, healthy tuberculosis contacts and non-contacts. Healthy tuberculin skin test (Mantoux) positive tuberculosis contacts demonstrated strong CTL response against RvCFP and Mantoux reactivity was found to correlate with CTL response. The specificity of CTL response in healthy Mantoux positive contacts was further assessed using different molecular weight fractions of RvCFP. Peripheral blood mononuclear cells (PBMCs) derived CTLs recognized multiple antigenic targets and demonstrated predominant cytotoxicity against low molecular weight (below 15 kDa) protein fractions as well as those migrated in the region of 30 kDa. Subsequently, evaluation of CTL responses against selected purified prominent T-cell antigens indicated maximum CTL response directed against Ag 85 complex proteins; most notably Ag 85 A. From this study, it is suggested that identification of more mycobacterial antigens activating various CTL subsets could be an important step for the rational designing of future antituberculous vaccine.

We report the role of human neutrophil peptide (HNP)-1 as an adjunct to antituberculosis (anti-TB) drugs. The combination of HNP-1, isoniazid, and rifampicin was evaluated against Mycobacterium tuberculosis H(37)Rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (MICs) of these agents. In vitro results revealed >1-log unit reductions even when HNP-1 and anti-TB drugs were used at 1/16 MICs. This combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 MICs of HNP-1 and drugs. HNP-1 used in conjunction with anti-TB drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. The effective therapeutic dosage of drugs could be reduced to half by supplementing HNP-1 in the therapeutic schedule. These results clearly suggest that HNP-1 can be used as adjunct chemotherapy with conventional drugs against TB., (Copyright 2004 Infectious Diseases Society of America)

Cholesterol-mediated mycobacteria entry into and survival within macrophages has added a new dimension to Tuberculosis research. The molecular mechanism through which cholesterol initiates this process is still poorly understood. The present study addressed to resolve this mechanism revealed that Mycobacterium tuberculosis possesses cholesterol-specific Receptor 'Ck'-like molecule responsible for mycobacterial entry into macrophages. Further human Receptor-Ck was found to regulate transcriptional expression of a gene that codes for Tryptophan-Aspartate containing coat (TACO) protein responsible for survival of mycobacteria within cells. Based upon these results, we propose that interaction of Receptor-Ck with cholesterol-rich membrane domains helps to create a 'Synaptic-junction' between mycobacteria and macrophage resulting in signalling events that are responsible for mycobacterium entry into and survival within macrophages.

The aim of the study was to investigate the activity of human neutrophil peptide (HNP)-1 to kill Mycobacterium tuberculosis H37Rv in vitro and ex vivo in the murine macrophage cell line J744A.1 on the basis of colony forming units. Macromolecular biosynthesis was studied by monitoring the incorporation of radioactive precursors into different macromolecules. The binding and localization studies were carried out with radioiodinated HNP-1 whereas the cytotoxicity of HNP-1 to macrophages was determined by trypan blue exclusion assay. A concentration dependent inhibition in the growth of M. tuberculosis H37Rv was observed in the presence of HNP-1. The minimum inhibitory concentration and median inhibitory concentration of HNP-1 were found to be 2.5 microg x mL(-1) and 0.8 microg x mL(-1). Treatment of both in vitro grown and phagocytosed mycobacterial cells with HNP-1 resulted in generalized inhibition in the macromolecular biosynthesis with maximum inhibition in deoxyribonucleic acid and lipid biosynthesis. HNP-1 exhibited equilibrium binding with respect to time and two-thirds of bound radioactivity was shown to be present inside the macrophages. Approximately 50% and 98% killing of intracellular mycobacteria was observed after 3 days of treatment with 5 microg x mL(-1) and 40 microg x mL(-1) of HNP-1, respectively. HNP-1 exhibited low cytotoxicity towards the macrophage cell line at the bactericidal concentration to mycobacteria. From the results of this study, it is concluded that human neutrophil peptide-1 possesses potent bactericidal activity against virulent mycobacteria in vitro as well as mycobacteria replicating within macrophages.

The immunoprotective behaviour of the 30-kDa secretory glycoprotein of Mycobacterium tuberculosis H37Rv has been investigated in different adjuvant systems in two mouse strains, NMR1 and C57BL/6J. In comparison with Freund's incomplete adjuvant (FIA) and dimethyldioctadecyl ammonium bromide (DDA), the 30-kDa glycoprotein complexed with polylactide-co-glycolide microparticles (PLG-MPs) induced maximum immunoreactivity in the two mouse strains. As compared with controls, immunisation with 30-kDa-PLG-MPs resulted in significantly greater protection in animals challenged with 1 x LD50 of M. tuberculosis H37Rv on the basis of survival rates and number of cfu in the infected organs 30 days after challenge. The degree of protection provided by 30-kDa-PLG-MPs was similar to that obtained with 30-kDa-FIA and higher than BCG immunisation. These findings suggest that biodegradable PLG microparticles can be used as an efficient carrier system for the key immunoprotective 30-kDa secretory protein antigen of M. tuberculosis H37Rv.

The biochemical mechanism of action of human neutrophil peptide-1 (HNP-1) against Mycobacterium tuberculosis H37Ra was studied. Mycobacteria grown in the presence of a subinhibitory concentration (IC50) of HNP-1 showed a significant decrease in the biosynthesis of vital macromolecules, as shown by the incorporation of various radiolabeled precursors. Mycobacterial cells grown in the presence of HNP-1 exhibited surface changes, as was evident from the increased number of binding sites for L-anilinonaphthalene 8-sulfonate. Permeability studies carried out with spheroplasts showed a significantly high permeability to a fluorescent probe, N-phenyl naphthylamine, in the presence of HNP-1. Significant changes in the cell wall and cell membrane were observed when HNP-1-grown cells were analysed by transmission electron microscopy. Our results suggest the mycobacterial cell wall/membrane to be the major target(s) of HNP-1.

The immunoreactivity of 70 kDa culture filtrate protein of Mycobacterium tuberculosis H37 Ra has been examined as such and by increasing its hydrophobicity through its conjugation to stearic acid ester (70 kDa-FAester). The cell mediated immune responses produced by 70 kDa-FAester encapsulated in phosphatidylcholine (PC) liposomes were significantly higher particularly at the 3rd week post immunization (wk p. im.) than that produced by the 70 kDa protein in PC liposomes, whereas humoral immune responses were non-significant. Further, these immune responses were comparable to that elicited by 70 kDa protein complexed with Freund's incomplete adjuvant (70 kDa-FIA). Results of this study suggest that changes in physicochemical nature of 70 kDa protein influences both the humoral and cellular immunoreactivity.

Six different secretory proteins of molecular weights (15, 26, 30, 41, 55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycobacterium tuberculosis H37Ra using different column chromatography techniques. These proteins were further examined for their ability to induce cell mediated (T-cell proliferation assay) and humoral immune response (ELISA) in mice immunized with total culture filtrate proteins. Out of six proteins, three proteins showed good reactivity. However, the activity was at a maximum with 30 kDa antigen. The immune response induced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FIA) was investigated and was found to be dose dependent. The T-cell response induced by this protein was skewed towards T-helper (Th1) cells as determined by the pronounced secretion of interleukin-2 (IL-2) and gamma-interferon (IFN-gamma). The protective activity of the 30 kDa protein was also evaluated and compared with reference to Bacillus Calmette Guerin (BCG) vaccine in the mice challenged with virulent M. tuberculosis H37Rv. The degree of protection afforded by the 30 kDa antigen on the basis of mortality and the significant decrease in c.f.u.'s recovered from different organs (lung, liver, spleen) after 30 days of challenge with LD50 of M. tuberculosis H37Rv was significantly higher in comparison to BCG vaccinated animals. However, the degree of immunity induced by this antigen decreased with time (when challenged 8 and 12 weeks post-immunization) but it was still comparable with BCG. These findings suggest that 30 kDa secretory protein of M. tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.

Objective To study the role of Interferon gamma release assay (IGRA) (QuantiFERON-TB Gold In Tube) in blood in the diagnostic work up of active tuberculosis (TB) in a high TB prevalence region. Design Prospective, comparative group study. Setting Subjects presenting to the services of the Pulmonary Medicine Department of a large tertiary care teaching hospital in northern India. Methods We prospectively enrolled, 30 cases of smear or histopathology proven newly diagnosed tuberculosis (18 pulmonary (PTB) and 12 extra-pulmonary (EPTB)) patients controls along with 30 healthy controls. All cases and controls underwent Tubercular Skin Test (TST) using 0.1 mL (1 tuberculin units) of purified protein derivative RT23 and IGRA using QuantiFERON-TB-Gold In Tube assay (QFT) in blood. For TSTan induration ≥10 mm was taken as positive. QFT testing was performed and interpreted as per manufacturer's (Cellestis) instructions. Results We studied 30 patients of active tuberculosis (18 PTB and 12 EPTB) and 30 healthy controls (14 men and 13 women, mean age 35.03±13.23 years). TST positivity had a sensitivity of 83.33% and 66.67% and specificity of 60% for both categories for the diagnosis of active PTB and EPTB respectively. In contrast QFT positivity had a sensitivity of 61.11% and 58.33% and specificity of 50% for the diagnosis of active PTB and EPTB respectively. Conclusions In this study the QFT-IGRA had a limited overall usefulness in the diagnosis of active pulmonary and extrapulmonary TB. QFT, thus can neither be taken as rule in nor rule out test in a high TB prevalence region like India. TST with all its limitation probably will continue to serve a useful purpose. Larger prospective studies are needed in high incidence countries to determine the sensitivity and specificity of IGRAs, their ability to serve as useful rule-out test for active TB. [ABSTRACT FROM AUTHOR]