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Precise and timely detection of tuberculosis (TB) is crucial to reduce transmission. This study aims to assess the accuracy of Xpert MTB/RIF Ultra on stool samples and systematically review the performance of Xpert MTB/RIF Ultra with different sample types by meta-analysis. Stool samples of smear-negative pulmonary TB (PTB), cervical lymph node TB, and abdominal TB patients were tested on the Xpert MTB/RIF Ultra system. Meta-analysis was performed on a set of 44 studies. Data were grouped by sample type, and the pooled sensitivity and specificity of Xpert MTB/RIF Ultra were calculated. The sensitivity of Xpert MTB/RIF Ultra with stool samples was 100% for smear-negative PTB, 27.27% for cervical lymph node TB, and 50% for abdominal TB patients, with 100% specificity for all included TB groups. The summary estimate for all PTB samples showed 84.2% sensitivity and 94.5% specificity, and EPTB samples showed 88.6% sensitivity and 96.4% specificity. Among all sample types included in our meta-analysis, urine showed the best performance for EPTB diagnosis. This pilot study supports the use of stool as an alternative non-invasive sample on Xpert MTB/RIF Ultra for rapid testing, suitable for both PTB and EPTB diagnosis.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant

Background: Rapidly growing mycobacteria (RGM) are increasingly being recognized as potential pathogens. RGM, particularly Mycobacterium abscessus, Mycobacterium fortuitum, and Mycobacterium chelonae, have been observed in both pulmonary and extrapulmonary infections including cutaneous, soft-tissue, and wound infections. However, there are limited reports of these potential pathogens from skin and soft-tissue infections. Moreover, the drug susceptibility profile of RGM is largely unknown in several regions of the world. Methods: We analyzed reports on RGM isolated from skin and soft-tissue infections globally for details of RGM species and drug susceptibility profile. We also analyzed the drug susceptibility profile of four RGM isolates, obtained from skin and soft-tissue infections in our laboratory, by broth microdilution method. Results: In the reports reviewed, the most common RGM isolated from skin and soft-tissue infections were M. abscessus (184/475, 38.7%), M. fortuitum (150/475, 31.5%), M. chelonae (72/475, 15%), and M. chelonae–M. abscessus complex (46/475, 9.6%). However, drug susceptibility was tested only in 26/39 (66.6%) reports. In our own laboratory, we obtained three isolates of M. abscessus and one isolate of M. fortuitum from one case of breast abscess and three cases of postsurgical wound infections. Maximum susceptibility of M. abscessus was observed to clarithromycin, amikacin, and linezolid. The M. fortuitum isolate was susceptible to clarithromycin, amikacin, clofazimine, and linezolid. Conclusion: Paucity of information available on RGM isolated from skin and soft-tissue infections highlights the need to be aware of the pathogenic potential and the drug susceptibility profile of these organisms.

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth indicator tube (MGIT), endorsed by the World Health Organization (WHO), is widely used. Further identification of a positive culture is done with the help of an immunochromatography assay, which often shows faint bands that are difficult to interpret. We analysed 125 BACTEC MGIT culture positive results, of which 11/16 (68.7%) of the doubtful assays, analysed by MGIT™ TBc Identification test (TBcId), were positive for Mycobacterium tuberculosis complex (MTBC), the remaining being non-tuberculous mycobacteria as determined by an in-house duplex polymerase chain reaction and line probe assay. Guidelines on faint or doubtful bands in immunochromatography assays are important so as not to overlook true-positive cases of TB.

Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5–5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5–93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-( N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of drug resistance necessitate the search for therapeutic vaccine strategies for tuberculosis (TB). Such strategies ought to elicit not only IFN-γ, but polyfunctional response including TNF-α, which is essential for protective granuloma formation. Here, we investigated the impact of PD-1 inhibition in facilitating protective polyfunctional T cells (PFTs), bacillary clearance, and disease resolution. We have observed PD-1 inhibition preferentially rescued the suppressed PFTs in active tuberculosis patients. In addition, polyfunctional cytokine milieu favored apoptosis of infected MDMs over necrosis with markedly reduced bacillary growth (≪CFU) in our in vitro monocyte-derived macrophages (MDMs) infection model. Furthermore, the animal study revealed a significant decline in the bacterial burden in the lungs and spleen of infected mice after in vivo administration of α-PD-1 along with antitubercular treatment. Our findings suggest that rescuing polyfunctional immune response by PD-1 inhibition works synergistically with antituberculosis chemotherapy to confer improved control over bacillary growth and dissemination. In summary, our data strongly indicate the therapeutic potential of α-PD-1 as adjunct immunotherapy that can rejuvenate suppressed host immunity and enhance the efficacy of candidate therapeutic vaccine(s).

A real-time immuno-PCR assay was deliberated to detect mycobacterial mannophosphoinositides (PIMs). A dynamic range of PIMs (0.9 pg/mL-10 ng/mL) was detected in TB patients, wherein 88.2% and 81.1% sensitivities were obtained in pulmonary TB and extrapulmonary TB respectively, with 96-96.4% specificity. This assay may translate into a diagnostic kit.

Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%–20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress. Methods: Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates. Results: The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates. Conclusion: The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress.

We report the draft genome sequence of Mycobacterium simiae, a slowly growing nontuberculous mycobacterium (NTM) isolated from a mouthwash sample of a healthy person. This genome of 6,603,693 bp exhibited a 66.13% GC content and 6,391 genes with 6,257 coding sequences, 3 rRNAs, and 78 tRNAs.

Background Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. Result By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase β gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. Conclusion The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

Additional file 4. Intergenic Variations identified in VPCI591 (Type II).

Additional file 5. Invariant genes in global clinical isolates (including VPCI591).

Additional file 6. Unique genic variations in VPCI591.

Additional file 7. Inter-genic variations unique to VPCI591.

Additional file 3. Intergenic Variations in VPCI591 (Type I).

In an attempt to uncover genotypic indicators for isoniazid (INH) resistance in M. tuberculosis, in addition to the canonical mutations in genes associated with INH resistance, including katG, inhA and fabG promoter; we analyzed, two INH monoresistant isolates, ASTS24/13 (INHR1) and SHR1/14 (INHR2). Targeted Sanger sequencing detected a canonical mutation at katG315 only in INHR2. Infection of THP-1 cells and exposure to antituberculosis drugs led to two-fold increase in the minimum inhibitory concentration of INH in INHR2. Whole genome sequences revealed that INHR1 and INHR2 belonged to Delhi Central Asian Strain and East African Indian lineages, respectively. The sequences were compared with INH susceptible isolates with the same lineage as the INH monoresistant strains. INHR1 had a novel unique mutation STOP420Trp in the efflux pump gene Rv0849, while INHR2 had a novel mutation Arg579Ser in efflux pump gene mmpL5. Comparison of lipid associated genes showed novel mutations in INHR1 in fadE16, fadD3 and fbpD; while INHR2 had mutations in fadE1, Rv0145, Rv1425, fadD9 and mmaA3. Both isolates also demonstrated novel mutations in cell wall associated genes. Our study highlights the importance of searching for alternate mechanisms of INH resistance that may contribute to the development of more comprehensive diagnostic tools.

Lipid metabolism forms the heart and soul of Mycobacterium tuberculosis life cycle. Starting from macrophage invasion at cholesterol rich micro-domains to a sustainable survival for infection by utilizing cholesterol, Mycobacterium displays the nexus of metabolic pathways around host derived lipids. mce4 operon acts as cholesterol import system in M. tuberculosis and here we demonstrate role of mce4A gene of this operon in cholesterol catabolism. Here M. tuberculosis H37Rv overexpressing Rv3499c (mce4A) recombinant was used as a model to decipher the metabolic flux during intake and utilization of host lipids by mycobacteria. We analysed the impact of mce4A expression on carbon shift initiated during cholesterol utilization necessary for long term survival of mycobacterium. Through transcriptional analysis, upregulation in methylcitrate cycle (MCC) and methylmalonyl pathway (MMP) genes was observed in Rv3499c overexpressing recombinants of M. tuberculosis H37Rv. Up-regulation of methylmalonyl pathway associated enzyme encoding genes increased accumulation of virulence associated mycobacterial lipids phthiocerol dimycocerates (PDIM) and sulfolipid (SL1). We demonstrate that MCC and MMP associated enzyme encoding genes are upregulated upon mce4A overexpression and lead to enhanced accumulation of PDIM and SL1 which are responsible for pathogenicity of M. tuberculosis.

Atypical pathogens including Mycoplasma pneumoniae and Legionella pneumophila are increasingly recognized as important causes of community-acquired pneumonia (CAP). Mycoplasma pneumoniae accounts for 20-40% of all CAP and L. pneumophila is responsible for 3-15% of cases. The paucity of data from India in this regard prompted us to conduct this prospective multicentric analysis to detect the prevalence of M. pneumoniae and L. pneumophila in our geographical region. A total of 453 patients with symptoms of pneumonia and 90 controls with no history of lower respiratory tract infections were included in the study. A duplex polymerase chain reaction (PCR) targeting 543 bp region of P1 adhesin gene of M. pneumoniae and 375 bp region of macrophage infectivity potentiator (mip) gene of L. pneumophila was standardized for simultaneous detection of these atypical pathogens. Respiratory secretions, blood, and urine samples were collected from each patient and control and were subjected to duplex PCR, culture and serology for M. pneumoniae and L. pneumophila. Urine samples were subjected for detecting L. pneumophila antigen. Among the 453 patients investigated for M. pneumoniae, 52 (11.4%) were positive for IgM antibodies, 17 were positive by culture, and seven tested positive by PCR (P1 gene). Similarly for L. pneumophila, 50 cases (11%) were serologically positive for IgM antibodies, one was positive by PCR (mip gene) and urine antigen detection. A total of eight samples were positive by duplex PCR for M. pneumoniae P1 gene (N = 7) and L. pneumophila mip gene (N = 1). Of the 90 controls, two samples (2.2%) showed IgM positivity, and 15 (16.7%) showed IgG positivity for M. pneumoniae. For L. pneumophila, three samples (3.3%) tested positive for IgM, and 12 (13.3%) tested positive for IgG antibodies. The study findings indicate the presence of M. pneumoniae and L. pneumophila in our geographical region, and a combination of laboratory approaches including PCR, culture, and serology is required for effective detection of these agents.

In the present study, IS6110 loop mediated isothermal amplification (LAMP) assay was modified using dUTP-UNG (uracil-DNA N-glycosylase) strategy to prevent carryover contamination, and was evaluated using clinical specimens. The clinical specimens were collected from 236 suspected patients of pulmonary tuberculosis and 315 specimens of suspected patients of extra pulmonary tuberculosis. DNA was extracted from specimens and used as template for nucleic acid amplification. The results were evaluated with culture method as gold standard. Modified IS6110 LAMP assay showed high sensitivity (94.4%) and specificity (97.2%) in specimens collected from suspected pulmonary tuberculosis patients. Sensitivity was comparatively less (86.67%) in extra pulmonary specimens while specificity was 94.04%. In conclusion, IS6110 LAMP assay was modified to prevent carry over contamination and it was validated to be rapid, sensitive and specific method with prospective application in resource-limited settings.

Purpose. We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. Methodology. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. Results. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. Conclusion. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

A complicated epidemiological situation characterized by significantly high tuberculosis (TB) morbidity is observed in West Siberia. This study was aimed to investigate the genetic characteristics of Mycobacterium tuberculosis circulating in the southern part of West Siberia (in the Omsk region).From March 2013 to January 2015, 100 isolates of M. tuberculosis were obtained from patients with pulmonary TB living in the Omsk region. Drug susceptibility testing was performed on Lowenstein-Jensen medium (absolute concentration method). Genetic typing of isolates was carried out by variable number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The genetic types and characteristics of cluster strains were determined using 15 MIRU-VNTR loci.Thirty six VNTR types were found. Twenty six (26.0%) isolates had a unique profile, and the remaining 74 were grouped in 10 clusters containing from 2 to 23 isolates. The Beijing genotype was found in 72 isolates, 61 (85.0%) of which were part of five clusters that included two large clusters containing 23 isolates. Other genetic families, such as Latin-American Mediterranean (LAM, 11.0%), S family (2.0%) and Haarlem (4.0%), were also detected. The genetic family of 11 isolates could not be determined. Six different VNTR profiles were found in these non-classified isolates. Only 16 per cent of isolates were sensitive to anti-TB drugs. The katG315 (94.8%) and rpoB531 (92.2%) mutations were identified in 77 multidrug-resistant M. tuberculosis isolates.This study showed that the M. tuberculosis population in the Omsk region was heterogeneous. The Beijing genotype predominated and was actively spreading. The findings obtained point to the need for the implementation of more effective preventive measures to stop the spread of drug-resistant M. tuberculosis strains.

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.

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.

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of drug resistance necessitate the search for therapeutic vaccine strategies for tuberculosis (TB). Such strategies ought to elicit not only IFN-γ, but polyfunctional response including TNF-α, which is essential for protective granuloma formation. Here, we investigated the impact of PD-1 inhibition in facilitating protective polyfunctional T cells (PFTs), bacillary clearance, and disease resolution. We have observed PD-1 inhibition preferentially rescued the suppressed PFTs in active tuberculosis patients. In addition, polyfunctional cytokine milieu favored apoptosis of infected MDMs over necrosis with markedly reduced bacillary growth (≪CFU) in our in vitro monocyte-derived macrophages (MDMs) infection model. Furthermore, the animal study revealed a significant decline in the bacterial burden in the lungs and spleen of infected mice after in vivo administration of α-PD-1 along with antitubercular treatment. Our findings suggest that rescuing polyfunctional immune response by PD-1 inhibition works synergistically with antituberculosis chemotherapy to confer improved control over bacillary growth and dissemination. In summary, our data strongly indicate the therapeutic potential of α-PD-1 as adjunct immunotherapy that can rejuvenate suppressed host immunity and enhance the efficacy of candidate therapeutic vaccine(s).

Aim: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). Methods: Real-time immuno-PCR (RT-I-PCR) assay was devised for the quantitative detection of a cocktail of mycobacterial MPT64 (Rv1980c) and PstS1 (Rv0934) in TB patients. Results: A broad dynamic range of 0.95 pg/ml–95 ng/ml of MPT64+PstS1 was detected in TB patients. In smear-positive (n = 59) and smear-negative (n = 42) pulmonary TB cases, sensitivities of 93.2 and 83.3% were observed, respectively with 92.8% specificity, whereas a sensitivity of 77.9% and a specificity of 91.3% were observed in extrapulmonary TB cases (n = 86). Furthermore, significantly reduced MPT64+PstS1 concentrations (p

Tuberculosis (TB) is one of the leading causes of death from a single infectious agent, and millions of people fall sick due to this disease every year. In spite of effective treatment, drug-resistant tuberculosis is a major problem worldwide. Globally, 3.5% of new cases and 18% of previously treated cases were estimated to have MDR-TB in 2017. Among these, 8.5% cases were estimated to have XDR-TB. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB are, thus, responsible for significant obstruction to global TB control. Drug resistance in M. tuberculosis primarily occurs through chromosomal mutations in genes encoding for drug target or drug-activating enzymes. However, alternative mechanisms, such as membrane impermeability, drug inactivation or modification by enzymes, and efflux pumps, may also be responsible for drug resistance. Insight into the mechanisms of drug resistance would help mankind in limiting the disease through the use and development of better diagnostic and therapeutic tools. Here we describe the prevalence of drug resistance in M. tuberculosis, the mechanisms of drug resistance, and the rapid diagnostic assays currently available to detect drug-resistant M. tuberculosis.

NTM are widely distributed in nature and have been isolated from soil, natural water, tap water, water used in showers and surgical solutions. Although Mycobacterium tuberculosis is by far the most important mycobacterial species from a public health perspective, other species of nontuberculous mycobacteria (NTM) are being encountered with increasing frequency and new species are being identified. While not all NTM are pathogenic for humans, many are. Pathogenic NTM are usually less virulent than M. tuberculosis, and potential pathogens may be isolated without obvious association with a disease. On the contrary, species usually considered benign, may produce disease especially in immune-compromised individuals. The prevalence of NTM among world population has significant geographic variation that might occur even within the same country. Because of the large number of potentially pathogenic NTM that can be encountered in the clinical laboratory, diagnosis of these organisms is required to ensure appropriate treatment. However, diagnosis of NTM infection is often difficult. Bacteriological, radiological and clinical criteria to diagnose a disease are often not able to discriminate between M. tuberculosis and NTM infection. Moreover, a positive tuberculin skin test, does not allow distinction between NTM and M. tuberculosis infection. Since, labour intensive conventional methods of speciation are not used in every laboratory, NTM infections go unreported and are thus ignored. A greater laboratorian-clinician coordination would greatly benefit the recognition and treatment of these potential pathogens.

Aims and objectives: Multidrug resistant TB (MDR-TB) and extensively drug resistant (XDR) are responsible for significant obstruction to global control of tuberculosis (TB). Drug resistance in Mycobacterium tuberculosis mainly occurs through chromosomal mutations in genes encoding for drug target or drug activating enzymes. However, alternative mechanisms, such as efflux pumps may also be responsible for drug resistance. Methods: Insight into the mechanisms of drug resistance would help in limiting the disease through the use and development of better diagnostic and therapeutic tools. The importance of rapid and effective methods of identification of drug resistance in M. tuberculosis cannot be over emphasized. Traditional laboratory techniques are time consuming and cumbersome and not sufficiently sensitive or specific to fight this menace. Results: Though rapid molecular methods such as GeneXpert and line probe assays are vital tools in the fight against drug resistant TB, major advances in next generation sequencing (NGS) technology are allowing increasingly rapid and accurate sequencing of entire bacterial genomes, providing unprecedented depth of information. Conclusion: NGS has the ability to cause a revolutionary paradigm shift in the diagnosis of drug resistant tuberculosis and in understanding the evolution of drug resistance. However, certain challenges need to be overcome to enable the use of this promising technology in routine diagnosis and research, especially in resource limited regions of the world.

There is need for rapid and cost-effective diagnostic test for tuberculosis. The present study was carried out to design a Loop-mediated isothermal amplification (LAMP) assay combined with lateral flow dipstick (LFD) as a point-of-care method for diagnosis of TB. LAMP assay targeting sdaA gene combined with LFD for sequence specific detection was standardized in user friendly and rapid format. It does not require sophisticated instruments and shows visual results instantly. The LAMP-LFD assay was validated using culture confirmed specimens. The assay was evaluated in a cross-sectional study using respiratory specimens collected from patients in Delhi, India and it showed high concordance with GeneXpert MTB/RIF assay. Lateral flow dipstick method has provided an excellent detection format with LAMP method. The LAMP-LFD assay showed high diagnostic accuracy in comparison to other methods and can be used as a point-of-care test in cost-effective manner.

To discover additional genotypic indicators for ethambutol (EMB) resistant M. tuberculosis, we studied polymorphisms in arabinofuranosyl transferase encoding genes aftA (Rv3792), aftB (Rv3805) and aftC (Rv2673) in 38 EMB resistant and 34 EMB susceptible isolates from India and a repository established by the World Health Organization (WHO) Special Programme for Research and Training in Tropical Disease (TDR) by DNA sequencing. The results were correlated with the minimum inhibitory concentration (MIC) of EMB and mutations in embB (Rv3795). The most common non-synonymous polymorphism identified in aftB was Asp397Gly in 12/38 (31.6%) EMB resistant and 3/34 (8.8%) EMB susceptible isolates. Interestingly, 10/12 (83.3%) EMB resistant isolates with aftB Asp397Gly mutation also carried embB306, embB402 or embB497 mutations. Association of Asp397Gly polymorphism with EMB resistance was statistically significant (p 0.0216). However, overexpression of the mutant aftB in M. tuberculosis H37Rv did not exhibit any change in the MIC. Whole genome sequencing of a panel of Indian isolates and SNP cluster grouping (SCG) of TDR strains revealed an association between aftB mutation Asp397Gly and Beijing genotype or SCG2, a cluster group representing the Beijing genotype. To conclude, though aftBAsp397Gly mutation is not associated with EMB resistance, this mutation may be a phylogenetic marker for the Beijing clade.