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Mycobacterium tuberculosis AND drug use and [keywords]
- Dielectrophoresis-based purification of antibiotic-treated bacterial subpopulations. [JOURNAL ARTICLE]
- Lab Chip 2014 Apr 23.
Persistence of bacteria during antibiotic therapy is a widespread phenomenon, of particular importance in refractory mycobacterial infections such as leprosy and tuberculosis. Persistence is characterized by the phenotypic tolerance of a subpopulation of bacterial cells to antibiotics. Characterization of these "persister" cells is often difficult due to the transient, non-heritable nature of the phenotype and due to the presence of contaminating material from non-persisting cells, which usually comprise the larger fraction. In this study, we use 3D carbon-electrode arrays for dielectrophoresis-based separation of intact cells from damaged cells, revealed by differential staining with propidium iodide, and we use this procedure to purify intact cells from cultures of Mycobacterium smegmatis treated with isoniazid, a frontline anti-tuberculosis drug. The method presented in this study could be used for rapid label-free enrichment of intact persister cells from antibiotic-treated cultures while preserving the metastable persister phenotype. This approach would facilitate the downstream analysis of low-frequency subpopulations of cells using conventional omics techniques, such as transcriptomic and proteomic analysis.
- High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes. [Journal Article]
- Sci Rep 2014.:4632.
Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19.
- Prediction of XDR/pre-XDR tuberculosis by genetic mutations among MDR cases from a hospital in Shandong, China. [JOURNAL ARTICLE]
- Tuberculosis (Edinb) 2014 Mar 29.
The prevalence of extensively drug-resistant (XDR) and pre-extensively drug-resistant (pre-XDR) tuberculosis in China highlights the need for rapid diagnosis. Molecular methods based on the detection of resistance-conferring mutations provide promising solution for rapid diagnosis. Here, we evaluated the accuracy of using mutations in gyrA, rrs and tlyA to predict resistance to levofloxacin (LEV), amikacin (AMK) and capreomycin (CAP), among 208 clinical multidrug-resistant (MDR) Mycobacterium tuberculosis strains collected in a local hospital in Shandong province, China. A total of 131 (63.0%, 131/208) strains were detected resistance to at least one of the 3-s line drugs by drug susceptible tests (DSTs). By comparing the mutation data with the phenotypic results, we found all mutations in three genes could specifically (with specificities from 93.9% to 100%) predict resistances with sensitivities of 77.8% for gyrA (LEV), 71.4% for rrs (AMK), 53.6% for rrs (CAP), 14.3% for tlyA (CAP), 64.3% for rrs and tlyA (CAP). The combination of these mutations could predict 68.9% and 63.4% pre-XDR and XDR TB, respectively. However, the positive predictive value of rrs for CAP resistance (57.7%) and the negative predictive values of gyrA for LEV resistance (74.5%) were not satisfying. Our results supported the use of genetic mutations to predict resistance to AMK and fluoroquinolones. An algorithm that combines molecular methods and traditional DST would be valuable for detecting resistance to second-line drugs in our hospital.
- Synthesis and Anti-tubercular screening of [(2-chloroquinolin-3-yl)methyl] thiocarbamide derivatives. [JOURNAL ARTICLE]
- Chem Biol Drug Des 2014 Apr 18.
A series of 1-(substituted-phenyl)-1-[(2-chloroquinolin-3-yl)methyl]thiocarbamide and 1-(substituted-phenyl)-1-[(2-chloroquinolin-3-yl)methyl]methylthiocarbamide derivatives was synthesized as anti-tubercular agent. The structure of quinolinyl amines and their thiocarbamide derivatives were established on the basis of IR, (1) H and (13) C-NMR and mass spectral data. All the compounds were tested in-vitro for antimycobacterial activity against Mycobacterium tuberculosis (ATCC-25177) in Lowenstein Jensen medium by well diffusion method and MIC by two fold serial dilution method. Results of the antitubercular screening revealed that compounds showed moderate to good antitubercular activity. Compound having two halogens in the phenyl rings viz. 3g, 3h, 4g and 4h exhibited MIC of 50 μg/mL. The computational parameters relevant to absorption and permeation of target compounds were also calculated and found to be well correlated with antitubercular activity. This article is protected by copyright. All rights reserved.
- Cyto-genotoxicity Assessment of Potential Anti-tubercular Drug Candidate Molecule-trans-cyclohexane-1, 4-diamine Derivative-9u in Human Lung Epithelial Cells A549. [Journal Article]
- Toxicol Int 2014 Jan; 21(1):69-77.
Increasing incidences of multiple drug-resistance (MDR) in Mycobacterium tuberculosis are emerging as one among the serious public health threats and socio-economic burden to the third world countries including India. Last couples of decades are witnesses of the dedicated and sustained efforts made toward the development of target specific and cost-effective antimicrobial agents against MDR-M. tuberculosis. However, the drugs in use are still incapable of controlling the upsurge of MDR. Thus, in order to address the issue, we synthesized a library of symmetrical trans-cyclohexane-1, 4-diamine derivatives and evaluated their anti-mycobacterium activity in H37RV strain of M. tuberculosis. A range of efficacy has been recorded in different derivatives of synthesized compounds and compound "9u" having i-propyl group substitution at p-position, was found to have more significant detrimental effects against the tested strain of M. tuberculosis. The present investigations were aimed to study whether the effective anti-mycobacterium concentrations of "9u" are biologically safe to human cells or not? The human lung epithelial cell line-A549 were exposed to a range of concentrations, i.e., at and above the anti-mycobacterium effective dose of "9u" for a period of 0-96 h. The standard endpoints of cytotoxicity viz., tetrazolium bromide salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), neutral red uptake, lactate dehydrogenase release, trypan blue dye exclusion assays; and genotoxicity viz., micronucleus and chromosomal aberrations assays were used to evaluate the bio-safety of test compound. The compound "9u" shows no significant cytotoxicity and genotoxicity in A549 cells exposed to 10(-5) M for 72 h, a concentration substantially higher than the concentration kill the H37Rv strain of M. tuberculosis. The compound 9u was found to be safe up to 10(-4) M if given for 24 h. The data reveal the therapeutic potential of compound 9u against M. tuberculosis without any having any cytotoxicity and genotoxicity responses.
- In vitro-in vivo activity relationship of substituted benzimidazole cell division inhibitors with activity against Mycobacterium tuberculosis. [JOURNAL ARTICLE]
- Tuberculosis (Edinb) 2014 Apr 1.
Structure based drug design was used to develop a compound library of novel 2,5,6- and 2,5,7-trisubstituted benzimidazoles. Three structural analogs, SB-P1G10, SB-P8B2 and SB-P3G2 were selected from this library for advanced study. In vitro studies revealed that SB-P8B2 and SB-P3G2 had sigmoidal kill-curves while in contrast SB-P1G10 showed a narrow zonal susceptibility. The in vitro studies also demonstrated that exposure to SB-P8B2 or SB-P3G2 was bactericidal, while SB-P1G10 treatment never resulted in complete killing. The dose curves for the three compounds against clinical isolates were comparable to their respective dose curves in the laboratory strain of Mycobacterium tuberculosis. SB-P8B2 and SB-P3G2 exhibited antibacterial activity against non-replicating bacilli under low oxygen conditions. SB-P3G2 and SB-P1G10 were assessed in acute short-term animal models of tuberculosis, which showed that SB-P3G2 demonstrated activity against M. tuberculosis. Together, these studies reveal an in vitro-in vivo relationship of the 2,5,6-trisubstituted benzimidazoles that serves as a criterion for advancing this class of cell division inhibitors into more resource intensive in vivo efficacy models such as the long-term murine model of tuberculosis and Pre-IND PK/PD studies. Specifically, these studies are the first demonstration of efficacy and an in vitro-in vivo activity relationship for 2,5,6-trisubstituted benzimidazoles. The in vivo activity presented in this manuscript substantiates this class of cell division inhibitors as having potency and efficacy against M. tuberculosis.
- First Evaluation of Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from Congo Revealed Misdetection of Fluoroquinolone Resistance by Line Probe Assay Due to a Double Substitution T80A-A90G in GyrA. [Journal Article]
- PLoS One 2014; 9(4):e95083.
Tuberculosis (TB) is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC) isolates collected in the two main cities of Congo.Over a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST). Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl) and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping.Of the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46%) were resistant to one or more first-line antiTB drugs. Of these, 15 (71%) were multidrug resistant (MDR). The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60%) in rpoB and S315T (87%) in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA.Taken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.
- Tubercular spondylitis in children. [REVIEW]
- Indian J Orthop 2014 3; 48(2):136-144.
Spine of the child has unique anatomy and growth potential to grow to adult size. Tuberculosis (TB) spine results in bone loss as well as disturbed growth potential, hence spinal deformities may progress as the child grows. The growth potential is also disturbed when the disease focus is surgically intervened. Surgery is indicated for complications such as deformity, neurological deficit, instability, huge abscess, diagnostic dilemma and in suspected drug resistance to mycobacterium tuberculosis. The child on antitubercular treatment needs to be periodically evaluated for weight gain and drug dosages need to be adjusted accordingly. The severe progressive kyphotic deformity should be surgically corrected. Mild to moderate cases should be followed up until maturity to observe progression/improvement of spinal deformity. The surgical correction of kyphotic deformity in active disease is less hazardous than in a healed kyphosis. The internal kyphectomy by extra pleural approach allows adequate removal of internal salient in paraplegic patients with healed kyphotic deformity.
- Surgical treatment of nontuberculous mycobacterial lung disease. [JOURNAL ARTICLE]
- Gen Thorac Cardiovasc Surg 2014 Apr 17.
While the prevalence of pulmonary tuberculosis has been decreasing, the prevalence of nontuberculous mycobacterial lung disease has been increasing. Unlike tuberculosis, nontuberculous mycobacterial disease is not communicable. However, their indolent nature may result in extensive parenchymal destruction, causing respiratory failure and vulnerability to airway infection. Nontuberculous mycobacterial lung disease, therefore, has been becoming a significant health problem. According to the 2007 American Thoracic Society/Infectious Diseases Society of America statement on nontuberculous mycobacterial diseases, the primary treatment is a multidrug treatment regimen. However, its efficacy is less than satisfactory for Mycobacterium avium complex lung disease, which is the most common type of nontuberculous mycobacterial lung diseases, and for Mycobacterium abscessus lung disease, which is notoriously resistant to chemotherapeutic drugs. The statement, therefore, has proposed a multidisciplinary treatment approach for these types of nontuberculous mycobacterial lung diseases: a combination of multidrug treatment regimen and adjuvant resectional surgery. This review covers the rationale, indication, procedure, and outcome of surgical treatment of nontuberculous mycobacterial lung disease. The rationale of surgery is to prevent disease progressing by removing the areas of lung most affected, harboring the largest amounts of mycobacteria. The indications for surgery include a poor response to drug therapy, the development of macrolide-resistant disease, or the presence of a significant disease-related complication such as hemoptysis. The surgical procedures of choice are various types of pulmonary resections, including wedge resection, segmentectomy, lobectomy, or pneumonectomy. The reported series have achieved favorable treatment outcomes in surgically treated patients with acceptable morbidity and mortality rates.
- Profiling of rpoB Mutations and MICs to Rifampicin and Rifabutin in Mycobacterium tuberculosis. [JOURNAL ARTICLE]
- J Clin Microbiol 2014 Apr 16.
Resistance to rifampicin (RIF) and rifabutin (RFB) in Mycobacterium tuberculosis is associated with mutations within an 81 bp region of the rpoB gene (RIF resistance-determining region, RRDR). Previous studies have shown that certain mutations in this region are more likely to confer high levels of RIF resistance, while others may be found in phenotypically susceptible isolates. In this study we sought to determine the relationship between MIC of RIF and RFB and rpoB-RRDR mutations in 32 multidrug resistant (MDR), 4 RIF mono-resistant and 5 susceptible M. tuberculosis clinical isolates. MICs were determined using the MGIT 960 system. Mutations in the rpoB-RRDR were determined by Sanger sequencing.S531L, S531W, H526Y, H526D and D516A-R529Q RpoB substitutions were associated with high MICs to RIF and RFB. Five isolates carrying the mutations L511P, H526L, H526N, and D516G/S522L were found to be susceptible to RIF. Several mutations were associated with resistance to RIF and susceptibility to RFB (F514FF, D516V, S522L). Whole genome sequencing of two MDR isolates without rpoB-RRDR mutations revealed a mutation outside of the RRDR (V146F, RIF MIC 50 μg/ml). The implications of polymorphisms identified in the second isolate and RIF resistance need to be further explored.Our study further establishes a correlation between mutations and MIC levels for RIF and also RFB in M. tuberculosis. Several rpoB mutations were identified in RIF and RFB susceptible isolates. The clinical significance of these findings requires further exploration. Until then, a combination of phenotypic and molecular testing for drug susceptibility testing is advisable.