SummaryTuberculosis (TB) is primarily associated with decline in immune health status. As gut microbiome (GM) is implicated in the regulation of host immunity and metabolism, here we investigate GM alteration in TB patients by 16S rRNA gene and whole-genome shotgun sequencing. The study group constituted of patients with pulmonary TB and their healthy household contacts as controls (HCs). Significant alteration of microbial taxonomic and functional capacity was observed in patients with active TB as compared to the HCs. We observed that Prevotella and Bifidobacterium abundance were associated with HCs, whereas butyrate and propionate-producing bacteria like Faecalibacterium, Roseburia, Eubacterium and Phascolarctobacterium were significantly enriched in TB patients. Functional analysis showed reduced biosynthesis of vitamins and amino acids in favour of enriched metabolism of butyrate and propionate in TB subjects. The TB subjects were also investigated during the course of treatment, to analyse the variation of GM. Although perturbation in microbial composition was still evident after a month's administration of anti-TB drugs, significant changes were observed in metagenome gene pool that pointed towards recovery in functional capacity. Therefore, the findings from this pilot study suggest that microbial dysbiosis may contribute to pathophysiology of TB by enhancing the anti-inflammatory milieu in the host.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus-like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS6110. The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5′ end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii. This study retracing the evolutionary events of HGT and IS6110-driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages. IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis, the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii, by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.
Background: Diagnosis of isolated mediastinal tuberculosis (TB) can be challenging. Endobronchial ultrasound (EBUS) increases the diagnostic yield by direct sonographic visualization of mediastinal and hilar lymph nodes. With the advent of molecular techniques such as Gene Xpert, their addition to the cytology and cultures increases the diagnostic yield and detection of rifampicin resistance (RR) which helps change the effective therapeutic regimen immediately. Materials and Methods: Prospective analysis of all patients undergoing EBUS-guided transbronchial needle aspiration (EBUS-TBNA) with a clinical possibility of TB in isolated mediastinal lymphadenopathy patients at a tertiary care referral center between June 2016 and January 2018. All patients had at least five passes from each node of which two passes from each lymph node sampled in 2 ml of saline for culture and Gene Xpert for microbiologic, pathologic, and molecular analysis as per hospital protocol. Results: Out of 60 patients, 44 were diagnosed to have mediastinal tuberculous lymphadenitis, 8 sarcoidosis, 2 malignancies, and 6 reactive lymphadenitis. TBNA cytology was positive in 40/44 patients (90.9%), out of which 18 patients were culture positive with the sensitivity of 100%, specificity 47.6%, positive predictive value (PPV) 45%, and negative predictive value (NPV) 100%, (P value 0.011). TBNA acid-fast bacilli (AFB) smear was positive in 20/44 patients (45.45%) out of which 12 were culture positive, with sensitivity of 67%, specificity 80.95%, PPV 60%, NPV 85% (P value 0.011). TBNA Gene Xpert was positive in 30/44 patients (68.2%), out of which 6 (13.63%) showed RR-TB and two were cytology negative. Sixteen patients where culture positive with sensitivity of 88.89%, specificity 66.67%, PPV 53.33%, NPV 93.33% (P value of 0.005). TBNA AFB culture was positive in 18/44 patients (40.9%). Conclusion: EBUS-TBNA is an effective and safe diagnostic tool for intrathoracic TB, especially for mediastinal tuberculous lymphadenitis. The combination of various tests increases the diagnostic yield. Mediastinal nodal aspirates traditionally believed to be paucibacillary can still be captured by Gene Xpert.
timely diagnosis of paucibacillary tuberculosis (tB) which includes smear-negative pulmonary tB (ptB) and extra-pulmonary tB (eptB) remains a challenge. this study was performed to assess the diagnostic utility of stool as a specimen of choice for detection of mycobacterial DnA in paucibacillary TB patients in a TB-endemic setting. Stool samples were collected from 246 subjects including 129 TB patients (62 PTB and 67 EPTB) recruited at TB hospital in Delhi, India. Diagnostic efficacy of stool IS6110 PCR (n = 228) was measured, using microbiologically/clinically confirmed TB as the reference standard. The clinical sensitivity of stool PCR was 97.22% (95% confidence interval (CI), 85.47-99.93) for detection of Mycobacterium tuberculosis in stool samples of smear-positive PTB patients and 76.92% (CI, 56.35-91.03) in samples from smear-negative PTB patients. Overall sensitivity of PCR for EPTB was 68.66% (CI, 56.16-79.44), with the highest sensitivity for stool samples from patients with lymph node TB (73.5%), followed by abdominal TB (66.7%) and pleural effusion (56.3%). Stool PCR presented a specificity of 95.12%. The receiver operating characteristic curve also indicated the diagnostic utility of stool PCR in TB detection (AUC: 0.882). The performance characteristic of the molecular assay suggests that stool DnA testing has clinical value in detection of tB.Tuberculosis (TB), a global health problem, is caused by Mycobacterium tuberculosis (Mtb) and has two ways of clinical manifestation. Apart from the common pulmonary TB (PTB) manifestation, extrapulmonary TB (EPTB) is identified as a major concern and contributes significantly to the disease burden worldwide. According to the World Health Organization (WHO), 10 million people fell ill with TB in 2018 1 . Although new strategies for diagnosis and treatment over the past decade have led to decline in death rate, it remains inadequate to reach the milestone of the WHO "End-TB" goal 1 . Apart from the diagnostic and treatment difficulties faced in the eradication of PTB, timely diagnosis and treatment of EPTB is considered a bigger challenge. This is due to the diverse, non-specific and paucibacillary presentation of EPTB at remote body sites and requirement of a longer treatment regimen 2 . In 2018, an increase in EPTB incidence was reported in India 3 , which is also considered as a TB high-burden country by the WHO. A probable explanation for this rise can be in part due to access to better diagnostic tools that highlight the fact that EPTB burden may have been underrepresented over the years. Thus, accurate and rapid diagnostic tests for PTB and EPTB are key to limit the spread of the epidemic.In India, diagnosis of TB is done by the Revised National Tuberculosis Control Program guidelines that follow the WHO recommendations ( Fig. 1). At present, although the diagnosis of PTB by sputum-smear direct microscopy is considerably established, the diagnosis of smear-negative PTB and EPTB poses serious challenges due to paucibacillary nature of the specimen. Diagnos...
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