Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 clinical isolates of M. tuberculosis from North India which are categorized as sensitive to first-line drugs, mono-resistant, multi-drug resistant and pre-extensively drug resistant isolates. WGS revealed that 20% of the isolates were co-infected with M. tuberculosis and non-tuberculous mycobacteria species. We identified 12,802 novel genetic variations in M. tuberculosis isolates including 343 novel SNVs in 38 genes which are known to be associated with drug resistance and are not currently used in the diagnostic kits for detection of drug resistant TB. We also identified M. tuberculosis lineage 3 to be predominant in the northern region of India. Additionally, several novel SNVs, which may potentially confer drug resistance were found to be enriched in the drug resistant isolates sampled. This study highlights the significance of employing WGS in diagnosis and for monitoring further development of MDR-TB strains.

Role of flexible bronchoscopy in patients with sputum-negative pulmonary tuberculosis.

BACKGROUND: Pulmonary tuberculosis is one of the major health concerns in the developing countries. Isolation of acid-fast bacilli (AFB) or tuberculosis bacilli from the sputum is required for the diagnosis. A proportion of suspected pulmonary tuberculosis (PTB) cases either clinically or radiologically will not produce sputum or will have sputum negative for AFB. These subsets of cases pose a diagnostic challenge to the treating clinicians. In this study, we present our experience and outcomes with flexible bronchoscopy in patients with sputum-negative pulmonary tuberculosis. MATERIALS AND METHODS: This was a prospective cross-sectional study, conducted at SDS Tuberculosis research Centre and Rajiv Gandhi Institute of Chest Diseases, Bengaluru, India, from 2010 to 2016. A total of 1095 flexible bronchoscopies were done during this period, out of which 180 were patients with sputum negative for AFB, but were strongly suspected to have pulmonary tuberculosis on clinical examination or radiologically. There were 106 males and 74 females. The age ranged between 11 and 68 years. All patients underwent complete evaluation of the tracheobronchial tree followed by bronchoalveolar lavage (BAL). Microbiological studies used were Ziehl-Neelsen (ZN) staining and culture in Lowenstein-Jensen (LJ) medium. Mucosal lesions suspected of tuberculosis were subjected to biopsy and histopathological confirmation. The data was analyzed. RESULTS: Out of 180 patients, 106 (58.88%) cases had positive AFB smear on BAL. The culture confirmed the diagnosis of pulmonary tuberculosis in 120 cases (66.66%). Histopathology showed caseous granuloma in 38 (42.22%) cases, nonspecific inflammation in 40 (44.44%) cases, and malignancy in 12 (13.33%) cases out of 90 cases, who underwent biopsy. There were no post-procedural complications. CONCLUSION: Bronchoscopy is the useful tool in the diagnosis of pulmonary tuberculosis in patients with sputum-negative pulmonary tuberculosis. It is also helpful in differentiating conditions having the clinical picture that mimics pulmonary tuberculosis.

Meta-analysis of host response networks identifies a common core in tuberculosis.

Tuberculosis remains a major global health challenge worldwide, causing more than a million deaths annually. To determine newer methods for detecting and combating the disease, it is necessary to characterise global host responses to infection. Several high throughput omics studies have provided a rich resource including a list of several genes differentially regulated in tuberculosis. An integrated analysis of these studies is necessary to identify a unified response to the infection. Such data integration is met with several challenges owing to platform dependency, patient heterogeneity, and variability in the extent of infection, resulting in little overlap among different datasets. Network-based approaches offer newer alternatives to integrate and compare diverse data. In this study, we describe a meta-analysis of host's whole blood transcriptomic profiles that were integrated into a genome-scale protein-protein interaction network to generate response networks in active tuberculosis, and monitor their behaviour over treatment. We report the emergence of a highly active common core in disease, showing partial reversals upon treatment. The core comprises 380 genes in which STAT1, phospholipid scramblase 1 (PLSCR1), C1QB, OAS1, GBP2 and PSMB9 are prominent hubs. This network captures the interplay between several biological processes including pro-inflammatory responses, apoptosis, complement signalling, cytoskeletal rearrangement, and enhanced cytokine and chemokine signalling. The common core is specific to tuberculosis, and was validated on an independent dataset from an Indian cohort. A network-based approach thus enables the identification of common regulators that characterise the molecular response to infection, providing a platform-independent foundation to leverage maximum insights from available clinical data.

Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

Proinflammatory chemokines are major mediators of exuberant immune response associated with Influenza A (H1N1) pdm09 virus infection.

In India, the case fatality ratio of the pandemic A (H1N1) pdm09 influenza was relatively higher when compared to seasonal Influenza A infection. Hypercytokinemia or "cytokine storm" has been previously implicated in the pathogenesis of other influenza viruses. The present study was undertaken to compare the cytokine profiles of A (H1N1) pdm09 influenza and seasonal H3 infection in Indian population and to correlate the findings with disease severity. Plasma levels of 18 cytokines/chemokines were measured by flow-cytometry using a bead based assay in patients infected with A (H1N1) pdm09 virus (n = 96) and Influenza A seasonal H3 virus (n = 30) categorised into mild, moderate, and severe groups along with healthy controls (n = 36). There was an overall trend indicating an exuberant cytokine/chemokine response in A (H1N1) pdm09 as compared to seasonal H3 influenza, which was more evident in severe cases, suggesting a role for these cytokines/chemokines in the pathogenesis of A(H1N1) pdm09. Increased levels of CXCL-8/IL-8, IL-10, IL-6, and IL-17A were seen in both A(H1N1) pdm09 influenza and seasonal H3 cases when compared to healthy controls. However, dysregulated production of proinflammatory chemokines was seen more pronounced in A (H1N1) pdm09 influenza cases as compared to seasonal H3 cases. This study has brought forth the potential role of chemokines as prognostic indicators of disease severity and outcome. Further research on modulating the host immune response to limit severity of the disease could help in the treatment and management of influenza.

Drug-Induced Hypothyroidism during Anti-Tuberculosis Treatment of Multidrug-Resistant Tuberculosis: Notes from the Field.

We followed 188 euthyroidic persons undergoing treatment for multidrug resistant tuberculosis (MDR-TB) in the state of Karnataka, India to determine the incidence of hypothyroidism during anti-tuberculosis treatment. Overall, among MDR-TB patients with valid thyroid stimulating hormone (TSH) values, about 23% developed hypothyroidism (TSH value ≥10 mIU/ml) during anti-tuberculosis treatment; the majority (74%) occurring after 3 months of treatment. Among 133 patients who received a regimen that contained ethionamide, 42 (32%) developed hypothyroidism. Among 17 patients that received a regimen that contained para-aminosalicylate sodium, 6 (35%) developed hypothyroidism. Among 9 HIV positive patients on anti-retroviral treatment, 4 (44%) developed hypothyroidism. These results differ from previously reported 4% incidence of hypothyroidism amongst patients who passively reported thyroidal symptoms during treatment, suggesting routine serologic monitoring of TSH throughout the course of treatment for MDR-TB is warranted.

Traumatic diaphragmatic hernia-our experience.

OBJECTIVE: To review our experience in the management of traumatic diaphragmatic hernia. MATERIALS AND METHODS: The records of all patients operated for diaphragmatic hernia between January 1998 and October 2008 at S.D.S Sanitorium and Rajiv Gandhi Institute of Chest Diseases, Bangalore, India were reviewed. Details of their clinical presentation, mode of diagnosis, operative findings and postoperative outcome were analysed. RESULTS: Twenty nine patients underwent surgery for traumatic diaphragmatic hernia. The cause of rupture was blunt trauma in 24(83%) patients and penetrating trauma in 5(17%) patients. In 21 (72%) patients the diagnosis was made within 24 hrs and in 8(28%) patients the diagnosis was made after 24 hrs. Thoracotomy was the most common surgical approach used in 20(69%) patients. Post operative morbidity was 24% and mortality was 13.8%. CONCLUSION: X-ray chest is still very useful in the diagnosis of diaphragmatic ruptures. Right sided ruptures are difficult to diagnose. Diaphragmatic hernia repair can be done through a thoracotomy with acceptable results in patients without concomitant intra abdominal injuries.