Mandira Basil - Academia.edu (original) (raw)

Papers by Mandira Basil

Molecular Microbiology, 2005

Little is known about the intracellular events that occur following the initial inhibition of Myc... more Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

Nontuberculous Mycobacteria (NTM)

Japanese journal of infectious diseases, 2010

We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for t... more We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for the early detection and identification of Mycobacterium tuberculosis directly from clinical samples. PCR-RFLP of hsp65 was applied to the DNA extracted directly from sputum samples (n = 226) collected from 226 patients. We could detect and identify M. tuberculosis in 84.5% of the acid-fast bacillus (AFB) smear-positive samples (n = 149) and 11% of the AFB smear-negative samples (n = 18) obtained from patients with clinical and radiological evidence of tuberculosis. Sputum samples (n = 59) obtained from patients suffering from respiratory diseases other than tuberculosis were included as negative controls. To test the sensitivity of the assay, we spiked a smear-negative sample with serial dilutions of H37Rv. The protocol could detect down to 10 organisms/microl. PCR-RFLP was found to be a simple and reproducible method for early detection of M. tuberculosis from sputum samples. The present...

Background: Rapidly growing mycobacteria (RGM) are increasingly being recognized as potential pat... more 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 (15...

The aim of this study was to ascertain the incidence of drug resistance of Mycobacterium tubercul... more The aim of this study was to ascertain the incidence of drug resistance of Mycobacterium tuberculosis isolates from patients in Delhi, India, being treated with DOTS and in private clinics, since a large proportion of patients with tuberculosis in India seek help from private healthcare sectors. Sputum samples were collected from 60 cases of tuberculosis attending a DOTS center and 42 patients from private clinics. Of these, 35 patients from the DOTS center and 12 patients from private clinics had a second sputum sample collected following two months of therapy. The isolated M. tuberculosis strains were assayed for isoniazid (INH), rifampicin (RIF), streptomycin (SM) and ethambutol (EMB) susceptibility by the proportion method. The frequencies of multidrug resistance (MDR) in the M. tuberculosis strains obtained from those treated with DOTS and in private centers were 12.7% and 5% (p > 0.5), respectively. Isolates obtained after two months of therapy showed a similar rate of MDR ...

Scientific Reports

Timely diagnosis of paucibacillary tuberculosis (TB) which includes smear-negative pulmonary TB (... more 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 wi...

Tropical Doctor

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth in... more 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.

Future Microbiology

Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts ... more 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...

SummaryM. tuberculosis is one of the most successful human pathogens causing tuberculosis that le... more SummaryM. tuberculosis is one of the most successful human pathogens causing tuberculosis that leads to highest daily morbidity worldwide. The evasion of the host immune responses is an important strategy that M. tuberculosis adopts. MprA (Rv0981), the response regulator of two component system is known for DNA binding activity in the pathogen and its role in persistent infection in the host. MprA is recognized as a late stage antigen during infection. A variant form of the protein MprA with G70S polymorphism (MprA*) is observed in one of our local and in several global clinical isolates of M. tuberculosis. Here we report the nuclear localization of MprA and MprA* in differentiated macrophages. MprA and MprA* increase the expression of TGF-β and IL-10, the immune suppressive cytokines in THP-1 derived macrophage cells. Concurrently the phago-lysosome fusion is significantly reduced as shown by infection with M.bovis BCG. We show that single nucleotide variation in clinical isolates ...

BackgroundGenome sequencing and genetic polymorphism analysis of clinical isolates of M.tuberculo... more BackgroundGenome 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.ResultBy sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M.tuberculosis, we identified 143 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 etham...

European Journal of Immunology

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of dr... more 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).

Indian Journal of Medical Research

International Journal of Mycobacteriology

Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%–20% of all cases of tuberculos... more 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.

Infection, Genetics and Evolution

Global burden of latent TB infection comprises one-third of the world population. Identifying pot... more Global burden of latent TB infection comprises one-third of the world population. Identifying potential Mycobacterium tuberculosis (Mtb) latency associated antigens that can generate protective immunity against the pathogen is crucial for designing an effective TB vaccine. Usually the immune system responds to a small number of amino acids as MHC Class I or Class II peptides. The precision to trigger epitope specific protective T-cell immune response could therefore be achieved with synthetic peptide-based subunit vaccine. In the present study we have considered an immunoinformatic approach using available softwares (ProPred, IEDB, NETMHC, BIMAS, Vaxijen2.0) and docking and visualizing softwares (CABSDOCK, HEX, Pymol, Discovery Studio) to select 10 peptides as latency antigens from 4 proteins (Rv2626, Rv2627, Rv2628, and Rv2032) of DosR regulon of Mtb. As Intracellular IFN-γ secreted by T cells is the most essential cytokine in Th1 mediated protective immunity, these peptides were verified as potential immunogenic epitopes in Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy contacts of TB patients (HTB) and 10 Category I Pulmonary TB patients (PTB).The antigen-specific CD4 and CD8 T cells expressing intracellular IFN-γ were analyzed using monoclonal antibodies in all subjects by multi-parameter flow cytometry. Both, PTB and HTB individuals responded to DosR peptides by showing increased frequency of IFN-γ+CD4 and IFN-γ+CD8 T cells. The T-cell responses were significantly higher in PTB patients in comparision to the HTB individuals. Additionally, our synthetic peptides and pools showed higher frequencies of IFN-γ+CD4 and IFN-γ+CD8 T cells than the peptides of Ag85B. This pilot study can be taken up further in larger sample size which may support the untapped opportunity of designing Mtb DosR inclusive peptide based post-exposure subunit vaccine.

Journal of Microbiological Methods

There is need for rapid and cost-effective diagnostic test for tuberculosis. The present study wa... more 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.

Tuberculosis

To discover additional genotypic indicators for ethambutol (EMB) resistant M. tuberculosis, we st... more 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.

Future Microbiology

Aim: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). Methods:... more 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

European journal of medicinal chemistry, Jan 25, 2018

A series of β-d-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-catalyzed cy... more A series of β-d-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-catalyzed cycloaddition reaction between azidosugar and 7-O-/7-alkynylated coumarins in 62-70% overall yields. The in vitro antimycobacterial activity evaluation of the synthesized triazolo-conjugates against Mycobacterium tuberculosis revealed that compounds were bactericidal in nature and some of them were found to be more active than one of the first line antimycobacterial drug ethambutol against sensitive reference strain H37Rv, and 7 to 420 times more active than all four first line antimycobacterial drugs (isoniazid, rifampicin, ethambutol and streptomycin) against multidrug resistant clinical isolate 591. Study of in silico pharmacokinetic profile indicated the drug like characters for the test molecules. Further, transmission electron microscopic experiments revealed that these compounds interfere with the constitution of bacterial cell wall possibly by targeting mycobacterial InhA and DNA g...

Tuberculosis (Edinburgh, Scotland), 2018

Mutations at embB306 are the most prevalent polymorphisms associated with ethambutol (EMB) resist... more Mutations at embB306 are the most prevalent polymorphisms associated with ethambutol (EMB) resistance, responsible for 40-60% of EMB resistant clinical cases of tuberculosis (TB). The present study analyzed additional mutations associated with EMB resistance in the embB, embC, embA and Rv3806c (ubiA) genes in 29 EMB resistant and 29 EMB susceptible clinical isolates of M. tuberculosis selected from 360 patients with TB. The entire ubiA gene, mutational hotspot regions of embB, embC, and upstream region of embA were screened for polymorphisms by DNA sequencing and the results correlated with minimum inhibitory concentrations (MIC) of EMB. The most common polymorphism identified in ubiA was at codon 149 (GAA to GAC), occurring in 5/29 (17.2%) resistant isolates and 7/29 (24%) susceptible isolates. Mutations in embB were most common at codon 306 (ATG to ATC/GTG), occurring only in EMB resistant isolates (20/29; 69%). Mutations in the upstream region of embA at -8, -11, -12 and -60 codo...

Molecular Microbiology, 2005

Little is known about the intracellular events that occur following the initial inhibition of Myc... more Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

Nontuberculous Mycobacteria (NTM)

Japanese journal of infectious diseases, 2010

We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for t... more We attempted to apply the PCR-restriction fragment length polymorphism (PCR-RFLP) technique for the early detection and identification of Mycobacterium tuberculosis directly from clinical samples. PCR-RFLP of hsp65 was applied to the DNA extracted directly from sputum samples (n = 226) collected from 226 patients. We could detect and identify M. tuberculosis in 84.5% of the acid-fast bacillus (AFB) smear-positive samples (n = 149) and 11% of the AFB smear-negative samples (n = 18) obtained from patients with clinical and radiological evidence of tuberculosis. Sputum samples (n = 59) obtained from patients suffering from respiratory diseases other than tuberculosis were included as negative controls. To test the sensitivity of the assay, we spiked a smear-negative sample with serial dilutions of H37Rv. The protocol could detect down to 10 organisms/microl. PCR-RFLP was found to be a simple and reproducible method for early detection of M. tuberculosis from sputum samples. The present...

Background: Rapidly growing mycobacteria (RGM) are increasingly being recognized as potential pat... more 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 (15...

The aim of this study was to ascertain the incidence of drug resistance of Mycobacterium tubercul... more The aim of this study was to ascertain the incidence of drug resistance of Mycobacterium tuberculosis isolates from patients in Delhi, India, being treated with DOTS and in private clinics, since a large proportion of patients with tuberculosis in India seek help from private healthcare sectors. Sputum samples were collected from 60 cases of tuberculosis attending a DOTS center and 42 patients from private clinics. Of these, 35 patients from the DOTS center and 12 patients from private clinics had a second sputum sample collected following two months of therapy. The isolated M. tuberculosis strains were assayed for isoniazid (INH), rifampicin (RIF), streptomycin (SM) and ethambutol (EMB) susceptibility by the proportion method. The frequencies of multidrug resistance (MDR) in the M. tuberculosis strains obtained from those treated with DOTS and in private centers were 12.7% and 5% (p > 0.5), respectively. Isolates obtained after two months of therapy showed a similar rate of MDR ...

Scientific Reports

Timely diagnosis of paucibacillary tuberculosis (TB) which includes smear-negative pulmonary TB (... more 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 wi...

Tropical Doctor

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth in... more 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.

Future Microbiology

Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts ... more 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...

SummaryM. tuberculosis is one of the most successful human pathogens causing tuberculosis that le... more SummaryM. tuberculosis is one of the most successful human pathogens causing tuberculosis that leads to highest daily morbidity worldwide. The evasion of the host immune responses is an important strategy that M. tuberculosis adopts. MprA (Rv0981), the response regulator of two component system is known for DNA binding activity in the pathogen and its role in persistent infection in the host. MprA is recognized as a late stage antigen during infection. A variant form of the protein MprA with G70S polymorphism (MprA*) is observed in one of our local and in several global clinical isolates of M. tuberculosis. Here we report the nuclear localization of MprA and MprA* in differentiated macrophages. MprA and MprA* increase the expression of TGF-β and IL-10, the immune suppressive cytokines in THP-1 derived macrophage cells. Concurrently the phago-lysosome fusion is significantly reduced as shown by infection with M.bovis BCG. We show that single nucleotide variation in clinical isolates ...

BackgroundGenome sequencing and genetic polymorphism analysis of clinical isolates of M.tuberculo... more BackgroundGenome 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.ResultBy sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M.tuberculosis, we identified 143 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 etham...

European Journal of Immunology

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of dr... more 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).

Indian Journal of Medical Research

International Journal of Mycobacteriology

Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%–20% of all cases of tuberculos... more 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.

Infection, Genetics and Evolution

Global burden of latent TB infection comprises one-third of the world population. Identifying pot... more Global burden of latent TB infection comprises one-third of the world population. Identifying potential Mycobacterium tuberculosis (Mtb) latency associated antigens that can generate protective immunity against the pathogen is crucial for designing an effective TB vaccine. Usually the immune system responds to a small number of amino acids as MHC Class I or Class II peptides. The precision to trigger epitope specific protective T-cell immune response could therefore be achieved with synthetic peptide-based subunit vaccine. In the present study we have considered an immunoinformatic approach using available softwares (ProPred, IEDB, NETMHC, BIMAS, Vaxijen2.0) and docking and visualizing softwares (CABSDOCK, HEX, Pymol, Discovery Studio) to select 10 peptides as latency antigens from 4 proteins (Rv2626, Rv2627, Rv2628, and Rv2032) of DosR regulon of Mtb. As Intracellular IFN-γ secreted by T cells is the most essential cytokine in Th1 mediated protective immunity, these peptides were verified as potential immunogenic epitopes in Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy contacts of TB patients (HTB) and 10 Category I Pulmonary TB patients (PTB).The antigen-specific CD4 and CD8 T cells expressing intracellular IFN-γ were analyzed using monoclonal antibodies in all subjects by multi-parameter flow cytometry. Both, PTB and HTB individuals responded to DosR peptides by showing increased frequency of IFN-γ+CD4 and IFN-γ+CD8 T cells. The T-cell responses were significantly higher in PTB patients in comparision to the HTB individuals. Additionally, our synthetic peptides and pools showed higher frequencies of IFN-γ+CD4 and IFN-γ+CD8 T cells than the peptides of Ag85B. This pilot study can be taken up further in larger sample size which may support the untapped opportunity of designing Mtb DosR inclusive peptide based post-exposure subunit vaccine.

Journal of Microbiological Methods

There is need for rapid and cost-effective diagnostic test for tuberculosis. The present study wa... more 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.

Tuberculosis

To discover additional genotypic indicators for ethambutol (EMB) resistant M. tuberculosis, we st... more 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.

Future Microbiology

Aim: There is an urgent need to design a reliable diagnostic test for tuberculosis (TB). Methods:... more 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

European journal of medicinal chemistry, Jan 25, 2018

A series of β-d-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-catalyzed cy... more A series of β-d-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-catalyzed cycloaddition reaction between azidosugar and 7-O-/7-alkynylated coumarins in 62-70% overall yields. The in vitro antimycobacterial activity evaluation of the synthesized triazolo-conjugates against Mycobacterium tuberculosis revealed that compounds were bactericidal in nature and some of them were found to be more active than one of the first line antimycobacterial drug ethambutol against sensitive reference strain H37Rv, and 7 to 420 times more active than all four first line antimycobacterial drugs (isoniazid, rifampicin, ethambutol and streptomycin) against multidrug resistant clinical isolate 591. Study of in silico pharmacokinetic profile indicated the drug like characters for the test molecules. Further, transmission electron microscopic experiments revealed that these compounds interfere with the constitution of bacterial cell wall possibly by targeting mycobacterial InhA and DNA g...

Tuberculosis (Edinburgh, Scotland), 2018

Mutations at embB306 are the most prevalent polymorphisms associated with ethambutol (EMB) resist... more Mutations at embB306 are the most prevalent polymorphisms associated with ethambutol (EMB) resistance, responsible for 40-60% of EMB resistant clinical cases of tuberculosis (TB). The present study analyzed additional mutations associated with EMB resistance in the embB, embC, embA and Rv3806c (ubiA) genes in 29 EMB resistant and 29 EMB susceptible clinical isolates of M. tuberculosis selected from 360 patients with TB. The entire ubiA gene, mutational hotspot regions of embB, embC, and upstream region of embA were screened for polymorphisms by DNA sequencing and the results correlated with minimum inhibitory concentrations (MIC) of EMB. The most common polymorphism identified in ubiA was at codon 149 (GAA to GAC), occurring in 5/29 (17.2%) resistant isolates and 7/29 (24%) susceptible isolates. Mutations in embB were most common at codon 306 (ATG to ATC/GTG), occurring only in EMB resistant isolates (20/29; 69%). Mutations in the upstream region of embA at -8, -11, -12 and -60 codo...