Clinico-pathological profile, radiological presentation and drug susceptibility pattern of new smear positive (category I) pulmonary tuberculosis: a single centre experience in Delhi, India

Background:Aim of current study was to determine the clinical characteristics, radiological, laboratory features and anti-tubercular drug sensitivity in new smear positive (category I) pulmonary tuberculosis cases in a tertiary care dedicated TB OPD, Delhi. Methods:The study was a cross-sectional observational study and consists of 100 cases of new smear positive pulmonary tuberculosis cases (category I) irrespective of age and sex. The sputum were collected, stained with Ziehl-Nielsen (Z-N) staining and ultimately inoculated on Lowenstein-Jensen (L-J) media for six weeks. All sputum smear positive cases were subjected to culture and drug-susceptibility testing by 1% proportion method on Lowenstein-Jensen (LJ) medium. The Drug-Susceptibility Testing (DST) for isoniazid (INH), rifampicin (R-cin), ethambutol (EMB) and streptomycin (SM) were performed.Results:The age & sex distribution of 100 patients showed that majority of the patients (79%) belonged to 2nd, 3rd and 4th decades & 60 % were males and 40% were female with male to female ratio 3:2 respectively. Cough (83%), fever (77%) and weight loss (76%) were the most common presenting clinical features. The chest X-ray of 100 smear positive patients showed that 53% of patients had evidence of 35% unilateral and 18%  bilateral consolidation and 46% had cavitary lesions on chest X-ray (PA view) with 37% and 9% of patients having unilateral and bilateral cavities respectively. Of these 82 culture positives, 56.1% (n=46) were susceptible to all first-line anti-tubercular drugs, while 43.9% (n=36) were resistant to mostly one or other anti-tubercular drugs (INH, R-cin, SM or EMB).  Conclusion: We stressed the importance of early diagnosis of new cases by clinico-pathological features, identifying of drug resistance trends in anti-tubercular treatment naïve patients, in order to assess the efficacy of current interventions. Overall, these findings emphasize the importance of early diagnosis of drug resistance pattern of M. tuberculosis isolates to anti-tubercular in category I patients as well as its association with HIV across the country to timely modify and strengthen the national programs in order to prevent the emergence of MDR-TB strains and avert the threat of XDR-TB.

Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

<p>Abstract</p> <p>Background</p> <p>The <it>mce </it>operons play an important role in the entry of <it>M. tuberculosis </it>into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of <it>mce </it>mutants. Recently, <it>mce1 </it>operon was found to extend over 13 genes, <it>fadD5 </it>(Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to <it>mce1 </it>among the four <it>mce </it>operons of <it>M.tuberculosis</it>. We have examined the function of this region.</p> <p>Results</p> <p>We predicted putative promoter activity of the 200 base pairs of non-coding, intergenic region between Rv0166 and Rv0167 <it>in silico </it>using MEME software and designate it as intergenic promoter, IGPr. We demonstrate both promoter activity and a putative negative regulatory function of this fragment by reporter assays carried out in the surrogate host <it>M.smegmatis</it>. We find that the repressive elements not only control the native promoter but also repress a heterologous promoter of <it>M.smegmatis</it>. The higher activity of the intergenic promoter in a clinical isolate in comparison with the wild type sequence from <it>M.tuberculosis </it>H37Rv could be correlated with a point mutation within the negative element. We have mapped two transcription start sites for <it>mce1 </it>operon both of which are utilized in <it>M.tuberculosis </it>H37Rv as well as the clinical isolate VPCI591. Our studies show that the promoter activity in the non-coding region is relevant not only in reporter gene expression but also in the expression of <it>mce1 </it>operon in <it>M. tuberculosis </it>cells grown in synthetic medium.</p> <p>Conclusion</p> <p>The <it>mce </it>operon of <it>M.tuberculosis </it>H37Rv potentially can be transcribed from two promoters P1 and P2, former mapping upstream of Rv0166 and the latter in the non-coding intergenic region between Rv0166 and Rv0167. The transcription initiation from P1 results in a transcript with Rv0166 while that from P2 will be without it. The sequences between the translation start site of Rv0167 and the promoter P2 have a negative regulatory role, as point mutation within the sequence leads to enhanced activity of P2 as well as a heterologous promoter from <it>M.smegmatis</it>. The mutation detected in the clinical isolate VPCI591 therefore behaves like a gain-of-function mutation.</p

Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach

Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains

<p>Abstract</p> <p>Background</p> <p>The presence of four mammalian cell entry (<it>mce</it>) operons in <it>Mycobacterium tuberculosis </it>suggests the essentiality of the functions of the genes in these operons. The differential expression of the four <it>mce </it>operons in different phases of <it>in vitro </it>growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.</p> <p>Here we investigate the extent of polymorphism in eight genes in the <it>mce1 </it>and <it>mce4 </it>operons of <it>M. tuberculosis </it>from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform.</p> <p>Results</p> <p>We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of <it>mce1 </it>and <it>mce4 </it>operons revealed that <it>yrbE1A </it>[<it>Rv0167</it>] was most polymorphic in <it>mce1 </it>operon while <it>yrbE4A </it>[<it>Rv3501c</it>] and <it>lprN </it>[<it>Rv3495c</it>] had the highest number of SNPs in the <it>mce4 </it>operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to <it>M. tuberculosis </it>using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation.</p> <p>Conclusions</p> <p>Our results show that SNPs in the coding sequences of <it>mce1 </it>and <it>mce4 </it>operons in clinical isolates can be significantly high. Moreover, <it>mce4 </it>operon is significantly more polymorphic than <it>mce1 </it>operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in <it>mce1 </it>operon and synonymous substitutions are more in <it>mce4 </it>operon. <it>In silico </it>modeling predict that nonsynonymous SNP at <it>mce1A </it>[<it>Rv0169</it>], a virulence gene could play a pivotal role in causing functional changes in <it>M. tuberculosis </it>that may reflect upon the biology of the bacteria.</p

Mycobacterium tuberculosis infected macrophages lead to apoptosis of antigen activated CD8 T cells

Aims and objectives: The role of CD4 T cells in immuno-pathogenesis of Mycobacterium tuberculosis (MTB) has been demonstrated in mouse models and in humans. However, the role of CD8 T cells in protective immunity against TB needs elucidation. Depletion of cytotoxic CD8 T cells have been found to be associated with reduced immunity to TB in different animal models. Apoptosis and hypo-responsiveness of T cells have been reported in TB patients and correlated with the persistence of infection. In a previous report, MTB specific T cells were observed, as well as bystander T cells’ apoptosis, which is induced by ex vivo infected autologous macrophages. It becomes relevant to find out the effect of infected macrophages on antigen-specific CD8 T cells during infection. Method: Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from 10 healthy BCG-vaccinated subjects. Monocytes from one aliquot of PBMC matured to macrophages for five days. These macrophages were infected with H37Rv at MOI of 1:10. In parallel, PBMC from a second aliquot were stimulated with CFP/WCL at a concentration of 5 μg/ml in the presence of IL-2 for five days. To study whether cytotoxic CD8 T cells undergo apoptosis in the presence of MTB-infected macrophages, CFP/WCL activated T cells were co-cultured with infected macrophages and uninfected macrophages for a further five days. The parameters studied were the expression of AnnexinV as an apoptosis marker, expression of CD95 to understand the role of Fas-FasL mediated apoptosis, expression of CD45/RO as a memory cell marker, expression of CD25 as activation markers on CD4 and CD8 T cells through flow cytometry at 48 h and 5 days of co-culture. The levels of IFN-γ and TNF-α were studied in the respective supernatant. Results: As compared with unactivated PBMC a significantly high percentage of CD4/CD25+ and CD8/CD25+ cells in infected Mφ-CFP/WCL stimulated T cell culture indicate the activation of T cells in response to MTB antigens. A significant percentage of CFP/WCL activated CD8 T cells were found to be AV positive in the infected co-culture assay at the fifth day indicating the apoptosis of antigen activated CD8 T cells. Interestingly, a significant percentage of CFP/WCL activated CD8/RO+ memory cells were found at 48 h of co-culture, but this percentage was reduced at the fifth day of co-culture. However, the CD4/RO cells percentage remained constant until the fifth day of co-culture. This reduction in CD8 /RO cells and the increase in CD8/AV+ cells suggested apoptosis of memory cells which may be induced by infected macrophages. A comparable percentage of CD8/CD95+ cells in infected and uninfected co-culture wells at both 48 h and the fifth day was observed. As compared with unactivated PBMC, the percentage of CD8/CD95+ cells was found to be significantly higher in both infected and uninfected co-culture assays, suggesting that observed increased apoptosis in infected co-culture assays was not mediated through Fas-FasL pathway. In the infected co-culture supernatant the IFN-γ concentration declined by the fifth day as compared with the uninfected co-culture; this can be correlated with reducing T cell number during infection. In contrast, a positive correlation was observed between the concentration of TNF-α and the percentage of CD8/AV+ cells by the fifth day in infected co-culture. It indicated the role of TNF-α released by MTB-infected macrophages in mediating CD8 T cell apoptosis. Conclusion: The present data indicates that MTB infection of macrophages could be responsible for apoptosis of CD8 T cells, which may be correlated with impaired immunity against TB

Differential signaling of inducible nitric oxide synthase induction in Mycobacterium tuberculosis infected alveolar epithelial cell line A549 in response to cytokines IFN-γ, TNF-α and IL-1β

Background: In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide (NO), but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-γ, TNF-α and IL-1β. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-κB pathways for the induction of inducible Nitric Oxide Synthase (iNOS) mRNA and NO production. Methods: Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Results: Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-γ or a combination of three cytokines (IFN-γ, TNF-α and IL-1β) formed DNA protein complexes with probes from both −5.2kb region (specific for binding of STAT-1 protein) and −5.8kb region (specific for binding of both STAT-1 and NF-κB) of the iNOS promoter. However, TNF-α or IL-1β stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from −5.2kb region. Conclusions: This differential response indicated that TNF-α/IL-1β does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-γ. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly

Comparative Genetic Association Analysis of Human Genetic Susceptibility to Pulmonary and Lymph Node Tuberculosis

Background: Tuberculosis (TB) manifests itself primarily in the lungs as pulmonary disease (PTB) and sometimes disseminates to other organs to cause extra-pulmonary TB, such as lymph node TB (LNTB). This study aimed to investigate the role of host genetic polymorphism in immunity related genes to find a genetic basis for such differences. Methods: Sixty-three, Single nucleotide polymorphisms (SNPs) in twenty-three, TB-immunity related genes including eleven innate immunity (SLCA11, VDR, TLR2, TLR4, TLR8, IRGM, P2RX7, LTA4H, SP110, DCSIGN and NOS2A) and twelve cytokine (TNFA, IFNG, IL2, Il12, IL18, IL1B, IL10, IL6, IL4, rs1794068, IL8 and TNFB) genes were investigated to find genetic associations in both PTB and LNTB as compared to healthy community controls. The serum cytokine levels were correlated for association with the genotypes. Results: PTB and LNTB showed differential genetic associations. The genetic variants in the cytokine genes (IFNG, IL12, IL4, TNFB and IL1RA and TLR2, 4 associated with PTB susceptibility and cytokine levels but not LNTB (p LTA4H, P2RX7, DCSIGN and SP110 showed susceptibility to LNTB and not PTB. Pathway analysis showed abundance of cytokine related variants for PTB and apoptosis related variants for LNTB. Conclusions: PTB and LNTB outcomes of TB infection have a genetic component and should be considered for any future functional studies or studies on susceptibility to pulmonary and extra-pulmonary TB

Role of Vitamins B, C, and D in the fight against tuberculosis

Worldwide, tuberculosis (TB) is still a serious and significant health concern, more so with the emergence of multidrug-resistant-TB. The inability of mankind to control this infection stems from the fact that the vaccines and drugs that were once effective against TB are no longer efficacious. This has led to a search for new antituberculous agents and adjuvant therapy. Vitamins are being revisited for their role in pathogenicity as well as for their antimycobacterial properties. Vitamins such as biotin and thiamin are essential for Mycobacterium tuberculosis and are required for establishment of infection. On the other hand, vitamins such as Vitamin C and Vitamin D have been shown to possess antimycobacterial properties. To combat M. tuberculosis, innovative strategies need to be devised, keeping in mind the efficacy of the agent to be used. Vitamins can prove to be useful agents capable of modifying the life cycle and biology of M. tuberculosis. We present here a brief overview of the available knowledge on thiamin, biotin, Vitamin C, and Vitamin D, keeping TB treatment and control in perspective