Direct Detection of Fluoroquinolone Resistance in Sputum Samples from Tuberculosis Patients by High Resolution Melt Curve Analysis.

Multidrug-resistant tuberculosis (MDR-TB) requires treatment with fluoroquinolone (FLQ) drugs, however, the excessive use of FLQ has led to the rise of extensively drug-resistant TB. In 2019, ~ 20% of total MDR-TB cases were estimated to be resistant to FLQ drugs. In the present study, we developed and evaluated the utility of high-resolution melt curve analysis (HRM) for the rapid detection of FLQ-resistant Mycobacterium tuberculosis for the first time directly from sputum samples. A reference plasmid library was generated for the most frequently observed mutations of gyrA gene and was used to discriminate between mutant and wild-type samples in the FLQ-HRM assay. The developed assay was evaluated on n = 25 MDR M. tuberculosis clinical isolates followed by validation on archived sputum DNA (n = 88) using DNA sequencing as a gold standard. The FLQ-HRM assay showed a 100% sensitivity [95% Confidence Interval (CI): 71.5 to 100] and specificity (95% CI: 39.7 to 100) in smear-positive category, and a sensitivity of 88.9% (95% CI: 77.3 to 95.8) with 84.2% (95% CI: 60.4 to 96.6) specificity in smear-negative category. The assay showed a high level of concordance of ~ 90% (κ = 0.74) with DNA sequencing, however, we were limited by the absence of phenotypic drug susceptibility testing data. In conclusion, HRM is a rapid, cost-effective (INR 150/USD 1.83) and closed-tube method for direct detection of FLQ resistance in sputum samples including direct smear-negative samples.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Direct Molecular Detection of Drug-Resistant Tuberculosis from Transported Bio-Safe Dried Sputum on Filter-Paper.

In 2019, amongst half a million new rifampicin-resistant tuberculosis (TB) cases, 78% were multi drug-resistant TB (MDR-TB). Access to rapid and Universal-Drug susceptibility testing (DST) to patients in remote areas is a major challenge to combat drug-resistant TB. To overcome this challenge, we had recently reported the development of 'TB Concentration & Transport kit' for bio-safe ambient temperature transport of dried sputum on filter-paper (Trans-Filter). The present study was conducted to evaluate the utility of DNA extracted from sputum on Trans-Filter in a Multiplex PCR-based sequencing assay (Mol-DSTseq) for diagnosing drug-resistant TB. The developed Mol-DSTseq assays were standardized on Mycobacterium tuberculosis clinical isolates (n = 98) and further validated on DNA extracted from sputum on Trans-Filter (n = 100). Using phenotypic DST as gold standard, the Mol-DSTseq assay showed 100% (95% Confidence Interval [CI] 79.4-100%) and 73.3% (95% CI 54.1-87.7%) sensitivity for detecting rifampicin and isoniazid resistance with a specificity of 85.1% (95% CI 66.2-95.8%) and 100% (95% CI:82.3-100%), respectively. For fluoroquinolones and aminoglycosides, the Mol-DSTseq assay showed a sensitivity of 78.5% (95% CI 49.2-95.3%) and 66.6% (95% CI 9.4-99.1%) with a specificity of 88.2% (95% CI 72.5-96.7%) and 100% (95% CI 93.1-100%), respectively. The Mol-DSTseq assays exhibited a high concordance of ~ 83-96% (κ value: 0.65-0.81) with phenotypic DST for all drugs. In conclusion, the 'TB Concentration and Transport kit' was compatible with Mol-DSTseq assays and has the potential to provide 'Universal-DST' to patients residing in distant areas in high burden countries, like India for early initiation of anti-tubercular treatment.

Functional insights into Mycobacterium tuberculosis DevR-dependent transcriptional machinery utilizing Escherichia coli.

DevR/DosR response regulator is believed to participate in virulence, dormancy adaptation and antibiotic tolerance mechanisms of Mycobacterium tuberculosis by regulating the expression of the dormancy regulon. We have previously shown that the interaction of DevR with RNA polymerase is essential for the expression of DevR-regulated genes. Here, we developed a M. tuberculosis-specific in vivo transcription system to enrich our understanding of DevR-RNA polymerase interaction. This in vivo assay involves co-transforming E. coli with two plasmids that express α, ß, ß' and σA subunits of M. tuberculosis RNA polymerase and a third plasmid that harbors a DevR expression cassette and a GFP reporter gene under the DevR-regulated fdxA promoter. We show that DevR-dependent transcription is sponsored exclusively by M. tuberculosis RNA polymerase and regulated by α and σA subunits of M. tuberculosis RNA polymerase. Using this E. coli triple plasmid system to express mutant variants of M. tuberculosis RNA polymerase, we identified E280 residue in C-terminal domain of α and K513 and R515 residues of σA to participate in DevR-dependent transcription. In silico modeling of a ternary complex of DevR, σA domain 4 and fdxA promoter suggest an interaction of Q505, R515 and K513 residues of σA with E178 and D172 residues of DevR and E471 of σA, respectively. These findings provide us with new insights into the interactions between DevR and RNA polymerase of M. tuberculosis which can be targeted for intercepting DevR function. Finally, we demonstrate the utility of this system for screening of anti-DevR compounds.

Phosphatase-defective DevS sensor kinase mutants permit constitutive expression of DevR-regulated dormancy genes in Mycobacterium tuberculosis.

The DevR-DevS/DosR-DosS two-component system of Mycobacterium tuberculosis, that comprises of DevS sensor kinase and DevR response regulator, is essential for bacterial adaptation to hypoxia by inducing dormancy regulon expression. The dominant phosphatase activity of DevS under aerobic conditions enables tight negative control, whereas its kinase function activates DevR under hypoxia to induce the dormancy regulon. A net balance in these opposing kinase and phosphatase activities of DevS calibrates the response output of DevR. To gain mechanistic insights into the kinase-phosphatase balance of DevS, we generated alanine substitution mutants of five residues located in DHp α1 helix of DevS, namely Phe-403, Gly-406, Leu-407, Gly-411 and His-415. For the first time, we have identified kinase positive phosphatase negative (K+P-) mutants in DevS by a single-site mutation in either Gly-406 or Leu-407. M. tuberculosis Gly-406A and Leu-407A mutant strains constitutively expressed the DevR regulon under aerobic conditions despite the presence of negative signal, oxygen. These mutant proteins exhibited ∼2-fold interaction defect with DevR. We conclude that Gly-406 and Leu-407 residues are individually essential for the phosphatase function of DevS. Our study provides new insights into the negative control mechanism of DevS by demonstrating the importance of an optimal interaction between DevR and DevS, and local changes associated with individual residues, Gly-406 and Leu-407, which mimic ligand-free DevS. These K+P- mutant strains are expected to facilitate the rapid aerobic screening of DevR antagonists in M. tuberculosis, thereby eliminating the requirement for hypoxic culture conditions.

Cognate sensor kinase-independent activation of Mycobacterium tuberculosis response regulator DevR (DosR) by acetyl phosphate: implications in anti-mycobacterial drug design.

The DevRS/DosT two-component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under 'non-inducing' aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase-phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate-grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild-type DevS and phosphorylation-defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous-like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate-dependent phosphorylation can occur in the absence of DevR's cognate kinases. Based on our findings, we conclude that anti-mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.

Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent Mycobacterium tuberculosis.

The DevR response regulator of Mycobacterium tuberculosis is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent M. tuberculosis H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the Rv3134c-devR-devS operon using M. tuberculosis H37Ra and H37Rv devR overexpression strains, designated as LIX48 and LIX50, respectively. Overexpression of DevR led to the up-regulation of a large number of DevR regulon genes in aerobic cultures of LIX48, but not in LIX50. To ascertain the involvement of PhoP response regulator, also known to co-regulate a subset of DevR regulon genes, we complemented the naturally occurring mutant phoPRa gene of LIX48 with the WT phoPRv gene. PhoPRv dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of devR expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoPRv to the Rv3134c promoter and further revealed that DevR and PhoPRv proteins exhibit differential DNA binding properties to the target DNA. Through co-incubations with DNA, ELISA, and protein complementation assays, we demonstrate that DevR forms a heterodimer with PhoPRv but not with the mutant PhoPRa protein. The study puts forward a new possible mechanism for coordinated expression of the dormancy regulon, having implications in growth adaptations critical for development of latency.

Multiple transcription factors co-regulate the Mycobacterium tuberculosis adaptation response to vitamin C.

BACKGROUND: Latent tuberculosis infection is attributed in part to the existence of Mycobacterium tuberculosis in a persistent non-replicating dormant state that is associated with tolerance to host defence mechanisms and antibiotics. We have recently reported that vitamin C treatment of M. tuberculosis triggers the rapid development of bacterial dormancy. Temporal genome-wide transcriptome analysis has revealed that vitamin C-induced dormancy is associated with a large-scale modulation of gene expression in M. tuberculosis. RESULTS: An updated transcriptional regulatory network of M.tuberculosis (Mtb-TRN) consisting of 178 regulators and 3432 target genes was constructed. The temporal transcriptome data generated in response to vitamin C was overlaid on the Mtb-TRN (vitamin C Mtb-TRN) to derive insights into the transcriptional regulatory features in vitamin C-adapted bacteria. Statistical analysis using Fisher's exact test predicted that 56 regulators play a central role in modulating genes which are involved in growth, respiration, metabolism and repair functions. Rv0348, DevR, MprA and RegX3 participate in a core temporal regulatory response during 0.25 h to 8 h of vitamin C treatment. Temporal network analysis further revealed Rv0348 to be the most prominent hub regulator with maximum interactions in the vitamin C Mtb-TRN. Experimental analysis revealed that Rv0348 and DevR proteins interact with each other, and this interaction results in an enhanced binding of DevR to its target promoter. These findings, together with the enhanced expression of devR and Rv0348 transcriptional regulators, indicate a second-level regulation of target genes through transcription factor- transcription factor interactions. CONCLUSIONS: Temporal regulatory analysis of the vitamin C Mtb-TRN revealed that there is involvement of multiple regulators during bacterial adaptation to dormancy. Our findings suggest that Rv0348 is a prominent hub regulator in the vitamin C model and large-scale modulation of gene expression is achieved through interactions of Rv0348 with other transcriptional regulators.

Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis.

BACKGROUND: A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model. RESULTS: Towards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells. CONCLUSIONS: It is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.

A novel aptamer-based test for the rapid and accurate diagnosis of pleural tuberculosis.

Pleural tuberculosis (pTB) is diagnosed by using a composite reference standard (CRS) since microbiological methods are grossly inadequate and an accurate diagnostic test remains an unmet need. The present study aimed to evaluate the utility of Mycobacterium tuberculosis (Mtb) antigen and DNA-based tests for pTB diagnosis. Patients were classified as 'Definite TB', 'Probable TB' and 'Non-TB' disease according to the CRS. We assessed the performance of in-house antigen detection assays, namely antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and aptamer-based Aptamer-Linked Immobilized Sorbent Assay (ALISA), targeting Mtb HspX protein and DNA-based tests namely, Xpert MTB/RIF and in-house devR-qPCR. ROC curves were generated for the combined group of 'Definite TB' and 'Probable TB' vs. 'Non-TB' disease group and cut-off values were derived to provide specificity of ≥98%. The sensitivity of ALISA was ∼93% vs. ∼24% of ELISA (p-value ≤0.0001). devR-qPCR exhibited a sensitivity of 50% vs. ∼22% of Xpert (p-value ≤0.01). This novel aptamer-based ALISA test surpasses the sensitivity criterion and matches the specificity requirement spelt out in the 'Target product profile' for extrapulmonary tuberculosis samples by Unitaid (Sensitivity ≥80%, Specificity 98%). The superior performance of the aptamer-based ALISA test indicates its translational potential to bridge the existing gap in pTB diagnosis.

Genome-wide expression profiling establishes novel modulatory roles of vitamin C in THP-1 human monocytic cell line.

BACKGROUND: Vitamin C (vit C) is an essential dietary nutrient, which is a potent antioxidant, a free radical scavenger and functions as a cofactor in many enzymatic reactions. Vit C is also considered to enhance the immune effector function of macrophages, which are regarded to be the first line of defence in response to any pathogen. The THP-1 cell line is widely used for studying macrophage functions and for analyzing host cell-pathogen interactions. RESULTS: We performed a genome-wide temporal gene expression and functional enrichment analysis of THP-1 cells treated with 100 µM of vit C, a physiologically relevant concentration of the vitamin. Modulatory effects of vitamin C on THP-1 cells were revealed by differential expression of genes starting from 8 h onwards. The number of differentially expressed genes peaked at the earliest time-point i.e. 8 h followed by temporal decline till 96 h. Further, functional enrichment analysis based on statistically stringent criteria revealed a gamut of functional responses, namely, 'Regulation of gene expression', 'Signal transduction', 'Cell cycle', 'Immune system process', 'cAMP metabolic process', 'Cholesterol transport' and 'Ion homeostasis'. A comparative analysis of vit C-mediated modulation of gene expression data in THP-1cells and human skin fibroblasts disclosed an overlap in certain functional processes such as 'Regulation of transcription', 'Cell cycle' and 'Extracellular matrix organization', and THP-1 specific responses, namely, 'Regulation of gene expression' and 'Ion homeostasis'. It was noteworthy that vit C modulated the 'Immune system' process throughout the time-course. CONCLUSIONS: This study reveals the genome-wide effects of physiological levels of vit C on THP-1 gene expression. The multitude of effects impacted by vit C in macrophages highlights its role in maintaining homeostasis of several cellular functions. This study provides a rational basis for the use of the Vitamin C- THP-1 cell model, to study biochemical and cellular responses to stresses, including infection with M. tuberculosis and other intracellular pathogens.

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis.

Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INH-inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses.

Mycobacterium tuberculosis (Mtb) owes its success as a pathogen in large measure to its ability to exist in a persistent state of 'dormancy' resulting in a lifelong latent tuberculosis (TB) infection. An understanding of bacterial adaptation during dormancy will help in devising approaches to counter latent TB infection. In vitro models have provided valuable insights into bacterial adaptation; however, they have limitations because they do not disclose the bacterial response to the intracellular environment wherein the bacteria are simultaneously exposed to multiple stresses. We describe the pleiotropic response of Mtb in the vitamin C (vit C) model of dormancy developed in our laboratory. Vit C mediates a rapid regulation of genes representing ~14 % of the genome in Mtb cultures. The upregulated genes were better represented in lipid, intermediary metabolism and regulatory protein categories. The downregulated genes mainly related to virulence, detoxification, information pathways and cell wall processes. A comparison of this response to that in other models indicates that vit C generates a multiple-stress environment for axenic Mtb cultures that resembles a macrophage-like environment. The bacterial response to vit C resembles responses to gaseous stresses such as hypoxia and nitric oxide, oxidative and nitrosative stresses, nutrient starvation and, notably, the activated macrophage environment itself. These responses demonstrate that the influence of vit C on Mtb gene expression extends well beyond the DevR dormancy regulon. A detailed characterization of the response to vit C is expected to disclose useful strategies to counter the adaptive mechanisms essential to Mtb dormancy.

Comprehensive insights into Mycobacterium tuberculosis DevR (DosR) regulon activation switch.

DevR regulon function is believed to be crucial for the survival of Mycobacterium tuberculosis during dormancy. In this study, we undertook a comprehensive analysis of the DevR regulon. All the regulon promoters were assigned to four classes based on the number of DevR binding sites (Dev boxes). A minimum of two boxes are essential for complete interaction and their tandem arrangement is an architectural hallmark at all promoters. Initial interaction of DevR with the conserved box is essential for its cooperative binding to adjacent sites bearing low to very poor sequence conservation and is the universal mechanism underlying DevR-mediated transcriptional induction. The functional importance of tandem arrangement was established by analyzing promoter variants harboring Dev boxes with altered spacing. Conserved sequence logos were generated from 47 binding sequences which included 24 newly discovered Dev boxes. In each half site of an 18-bp binding motif, G(5) and C(7) are essential for DevR binding. Finally, we show that DevR regulon induction occurs in a temporal manner and genes that are induced early are also usually powerfully induced. The information theory-based approach along with binding and temporal expression studies provide us with comprehensive insights into the complex pattern of DevR regulon activation.

DNA Aptamer Targets Mycobacterium tuberculosis DevR/DosR Response Regulator Function by Inhibiting Its Dimerization and DNA Binding Activity.

Tuberculosis is recognized as one of the major public health threats worldwide. The DevR-DevS (DosR/DosS) two-component system is considered a novel drug target in Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, owing to its central role in bacterial adaptation and long-term persistence. An increase in DevR levels and the decreased permeability of the mycobacterial cell wall during hypoxia-associated dormancy pose formidable challenges to the development of anti-DevR compounds. Using an in vitro evolution approach of Systematic Evolution of Ligands by EXponential enrichment (SELEX), we developed a panel of single-stranded DNA aptamers that interacted with Mtb DevR protein in solid-phase binding assays. The best-performing aptamer, APT-6, forms a G-quadruplex structure and inhibits DevR-dependent transcription in Mycobacterium smegmatis. Mechanistic studies indicate that APT-6 functions by inhibiting the dimerization and DNA binding activity of DevR protein. In silico studies reveal that APT-6 interacts majorly with C-terminal domain residues that participate in DNA binding and formation of active dimer species of DevR. To the best of our knowledge, this is the first report of a DNA aptamer that inhibits the function of a cytosolic bacterial response regulator. By inhibiting the dimerization of DevR, APT-6 targets an essential step in the DevR activation mechanism, and therefore, it has the potential to universally block the expression of DevR-regulated genes for intercepting dormancy pathways in mycobacteria. These findings also pave the way for exploring aptamer-based approaches to design and develop potent inhibitors against intracellular proteins of various bacterial pathogens of global concern.

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study.

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as 'Definite' TBM (n = 8), 'Probable' TBM (n = 15), 'Possible' TBM (n = 21)] and 'Non-TBM' group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of 'Definite' TBM and 'Non-TBM' category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1-100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8-69.6) for EV-DNA and 77.3% (95%CI:62.1-88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2-91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.

Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis.

The diagnosis of abdominal tuberculosis (aTB) is challenging and there is an urgent need for an accurate diagnostic test. We have developed a high affinity DNA aptamer against GlcB antigen of Mycobacterium tuberculosis (Mtb). We further compared the diagnostic utility of in-house-generated high affinity DNA aptamers and polyclonal antibodies against two Mtb antigens, namely GlcB and HspX, in ascitic fluid samples. These diagnostic reagents were assessed in patients (n = 94) who were categorized as 'Definite TB', 'Probable TB', 'Possible TB' (taken together as aTB) and 'Non-TB' disease. Receiver operating characteristic curves were used to derive cut-off values to provide ≥93% specificity. Aptamer Linked Immobilized Sorbent Assay (ALISA) for HspX and GlcB exhibited a sensitivity of ∼84% and 50%, respectively (p-value <0.01). In contrast, antibody-based ELISA exhibited a lower sensitivity of ∼18% and ∼28% for HspX and GlcB, respectively (p-value <0.0001 and p = 0.05 for HspX and GlcB ELISA vs. ALISA, respectively). HspX ALISA detected 32/38 aTB cases, while Xpert detected only 9 samples. In conclusion, HspX aptamer-based test was found to be superior to the other tests for diagnosing aTB and it nearly fulfils the sensitivity criteria of WHO's 'Target Product Profile' for extrapulmonary tuberculosis (sensitivity ≥80%, specificity 98%).

The residue threonine 82 of DevR (DosR) is essential for DevR activation and function in Mycobacterium tuberculosis despite its atypical location.

The DevR (DosR) response regulator initiates the bacterial adaptive response to a variety of signals, including hypoxia in in vitro models of dormancy. Its receiver domain works as a phosphorylation-mediated switch to activate the DNA binding property of its output domain. Receiver domains are characterized by the presence of several highly conserved residues, and these sequence features correlate with structure and hence function. In response regulators, interaction of phosphorylated aspartic acid at the active site with the conserved threonine is believed to be crucial for phosphorylation-mediated conformational change. DevR contains all the conserved residues, but the structure of its receiver domain in the unphosphorylated protein is strikingly different, and key threonine (T82), tyrosine (Y101), and lysine (K104) residues are placed uncharacteristically far from the D54 phosphorylation site. In view of the atypical location of T82 in DevR, the present study aimed to examine the importance of this residue in the activation mechanism. Mycobacterium tuberculosis expressing a DevR T82A mutant protein is defective in autoregulation and supports hypoxic induction of the DevR regulon only very weakly. These defects are ascribed to slow and partial phosphorylation and the failure of T82A mutant protein to bind cooperatively with DNA. Our results indicate that the T82 residue is crucial in implementing conformational changes in DevR that are essential for cooperative binding and for subsequent gene activation. We propose that the function of the T82 residue in the activation mechanism of DevR is conserved in spite of the unusual architecture of its receiver domain.

Evaluation of 'TBDetect' sputum microscopy kit for improved detection of Mycobacterium tuberculosis: a multi-centric validation study.

OBJECTIVES: The present study aimed to evaluate the performance of the 'TBDetect' kit-based bio-safe fluorescent microscopy filter (BioFM-Filter) microscopy in comparison with direct smear microscopy and culture for the detection of pulmonary tuberculosis (TB) in a multi-centric setting in India. METHODS: The TBDetect kit enables sputum concentration through filtration using the BioFM-Filter for improved and bio-safe smear microscopy. We evaluated the performance of the TBDetect kit in a six-site multi-centric validation study on sputum collected from 2086 presumptive TB patients. RESULTS: The combined positivity of TBDetect microscopy performed on these sputum samples was 20% (n = 417/2086) vs 16.1% of light-emitting diode fluorescence microscopy (LED-FM, n = 337/2086) and 16% of Ziehl Neelsen (ZN) smear microscopy (n = 333/2086). The increment in positivity of TBDetect over both LED-FM and ZN smears was significant (p < 0.001). The overall sensitivity of TBDetect for six sites was ~55% (202/367, 95% confidence interval (CI): 50, 60%) vs 52% (191/367, 95% CI: 47, 57%) for LED-FM (p 0.14) and 50.9% (187/367, 95% CI: 46, 56%) for ZN smear (p < 0.05), using Mycobacterium Growth Indicator Tube culture (MGIT, n = 1949, culture positive, n = 367) as the reference standard. A bio-safety evaluation at six sites confirmed efficient sputum disinfection by TBDetect; 99.95% samples (1873/1874) were sterile after 42 days of incubation. Scientists and technicians at the study sites indicated the ease of use and convenience of TBDetect microscopy during feedback. CONCLUSIONS: TBDetect added value to the smear microscopy test due to its improved performance, convenience and user safety. These findings indicate that equipment-free TBDetect technology has the potential to improve TB diagnosis in basic laboratory settings by leveraging on the existing nationwide network of designated microscopy centres and primary healthcare centres.

Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis.

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of ≤16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.