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Mycobacterium tuberculosis AND drug use and [keywords]
- Finer characterization of Mycobacterium tuberculosis using spoligotyping and 15-loci MIRU-VNTRs reveals phylogeographical specificities of isolates circulating in Guyana and Suriname. [JOURNAL ARTICLE]
- Infect Genet Evol 2014 Dec 17.
In this study we used spoligotyping and 15-loci MIRU-VNTRs for a finer characterization of Mycobacterium tuberculosis strains isolated from patients residing in Guyana (n=74) and Suriname (n=80). The mean age of the patients was 38.5 years (36.5 and 40.2 years for Guyana vs. Suriname), with a male-to-female sex-ratio of 3.11 (2.25 and 2.27 for Guyana vs. Suriname). Spoligotyping and 15-loci MIRU-VNTRs led to a total of 41 and 65 different patterns respectively, with an overall clustering rate of 83.8% vs. 68.8%. Combined spoligotyping and VNTR analysis led to the detection of 18 clusters of 2-41 isolates, with an overall clustering of 67.5% and a recent "n-1" transmission rate of 55.8%. Importantly, Guyana was characterized by a significantly higher percentage of clustered isolates than Suriname (79.7% vs. 56.3%; p=0.0019). Three big spoligo/MIRU (SIT/MIT) clusters containing >10 isolates each were shared between the 2 countries, and concerned: T1 sublineage cluster 53/861 (n=41, 34 in Guyana vs. 4 in Suriname); EAI6-BGD1 sublineage cluster 1340/860 (n=16, 3 in Guyana vs. 13 in Suriname); and T1 sublineage cluster 131/146 (n=11, 6 in Guyana vs. 5 in Suriname); as well as 2 smaller clusters of 2 and 3 isolates respectively. However, the relative phylogeographical specificities of strains in circulation as well as a lack of drug-resistance observed among strains from Suriname suggested that trans-border transmission of drug-resistant isolates occurred less frequently than thought. Tracing and interrupting transmission channels of a specific clone (SIT53/15-MIT861) should become a priority in Guyana, not only because it is by far most abundant but also because it accounts for almost half of the drug resistant isolates (n=8/17, 47.1%) in our study, and clustered 5/12 (41.7%) MDR isolates.
- Frequency of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis Strains Isolated from Patients in West of Iran. [JOURNAL ARTICLE]
- Microb Drug Resist 2014 Dec 19.
Background and Objective: Tuberculosis (TB) is a devastating infectious disease causing high mortality and morbidity worldwide. The most serious threat related to tuberculosis control is the recent emergence of drug-resistant tuberculosis strains. The aim of the present study was to identify various types of mutations in the rpoB region in rifampicin-resistant strains isolated from sputum of tuberculosis patients. Materials and Methods: Drug susceptibility of 125 Mycobacterium tuberculosis isolates was determined using the CDC standard conventional proportional method. Target DNA of M. tuberculosis was amplified by polymerase chain reaction, hybridized, and scanned. We used the low cost density array (LCD-array) to detect mutations within the 90-bp rpoB region. Each array is a transparent, prestructured polymer support using a nonfluorescent detection principle based on the precipitation of a clearly visible dark substrate. Results: Of the 125 M. tuberculosis isolates, 35 (28%) were found to be rifampicin-resistant and using the LCD array revealed point mutations at nine different codons as follows: S512T (AGC→ACC) (20%), D516V (GAC→GTC) (20%), H526D (CAC→GAC) (6%), H526R (CAC→CGC) (20%), H526Y (CAC→TAC) (23%), and S531W (TCG→TGG) (8%), and the most frequent site mutations were L511P (CCG→CTG) (46%), followed by S531l (TCG→TTG) (40%) and D516Y (GAC→TAC) (26%). Conclusion: Our data significantly differs from previously reported mutation frequencies for codon 526 (CAC to GAC) among Italian isolates (40.1%) and Greek isolates (17.6%). Phenotypic testing is time-consuming and requires laboratory resources. Microarray rpoB is useful in detecting rifampicin resistance-determining region-associated site mutations of rifampicin-resistant M. tuberculosis isolates.
- Revisiting Anti-tuberculosis Activity of Pyrazinamide in Mice. [JOURNAL ARTICLE]
- Mycobact Dis 2014 May 5.:145.
The mechanism of action of pyrazinamide, a key sterilizing drug in the treatment of tuberculosis, remains elusive; pyrazinamide is a pro-drug that requires activation by a bacterial-encoded enzyme, and its activity is most apparent on non-replicating Mycobacterium tuberculosis. Recently, it has been suggested that pyrazinamide might exert also some host-directed effect in addition to its antimicrobial activity. To address this possibility, three sequential experiments were conducted in immune-competent BALB/c and in immune-deficient, athymic nude mice. In the first experiment, BALB/c mice infected with M. bovis, which is naturally resistant to pyrazinamide because it is unable to activate the drug, were treated with standard drug regimens with and without pyrazinamide to specifically detect a host-directed effect. As no effect was observed, pyrazinamide activity was compared in M. tuberculosis-infected BALB/c and nude mice to determine whether the effect of pyrazinamide would be reduced in the immune deficient mice. As pyrazinamide did not appear to have any affect in the nude mice, a third experiment was performed in which rifampin was replaced with rifapentine (a similar drug with a longer half-life) to permanently suppress mycobacterial growth. In these experimental conditions, the antimicrobial effect of pyrazinamide was clear. Therefore, the results of our studies rule out a significant host-directed effect of pyrazinamide in the TB infected host.
- The Mycobacterium tuberculosis Uganda II family and resistance to first-line anti-tuberculosis drugs in Uganda. [JOURNAL ARTICLE]
- BMC Infect Dis 2014 Dec 19; 14(1):703.
BackgroundThe global increase in the burden of multidrug-resistant tuberculosis (MDR-TB) underscores an urgent need for data on factors involved in generation and spread of TB drug resistance. We performed molecular analyses on a representative sample of Mycobacterium tuberculosis (MTB) isolates. Basing on findings of the molecular epidemiological study in Kampala, we hypothesized that the predominant MTB strain lineage in Uganda is negatively associated with anti-TB drug resistance and we set out to test this hypothesis.MethodsWe extracted DNA from mycobacterial isolates collected from smear-positive TB patients in the national TB drug resistance survey and carried out IS6110-PCR. To identify MTB lineages/sub lineages RT-PCR SNP was performed using specific primers and hybridization probes and the `melting curve¿ analysis was done to distinguish the Uganda II family from other MTB families. The primary outcome was the distribution of the Uganda II family and its associations with anti-TB drug resistance and HIV infection.ResultsOut of the 1537 patients enrolled, MTB isolates for 1001 patients were available for SNP analysis for identification of Uganda II family, of which 973 (97%) had conclusive RT-PCR results. Of these 422 (43.4%) were of the Uganda II family, mostly distributed in the south west zone (55.0%; OR¿=¿4.6 for comparison with other zones; 95% CI 2.83-7.57; p¿<¿0.001) but occurred in each of the other seven geographic zones at varying levels. Compared to the Uganda II family, other genotypes as a group were more likely to be resistant to any anti-TB drug (ORadj =2.9; 95% CI 1.63-5.06; p¿=¿0.001) or MDR (ORadj 4.9; 95% CI, 1.15-20.60; p¿=¿0.032), even after adjusting for geographic zone, patient category, sex, residence and HIV status. It was commonest in the 25¿34 year age group 159/330 (48.2%). No association was observed between Uganda II family and HIV infection.ConclusionThe Uganda II family is a major cause of morbidity due to TB in all NTLP zones in Uganda. It is less likely to be resistant to anti-TB drugs than other MTB strain lineages.
- Correlating Minimum Inhibitory Concentrations of ofloxacin and moxifloxacin with gyrA mutations using the genotype MTBDRsl assay. [JOURNAL ARTICLE]
- Tuberculosis (Edinb) 2014 Dec 3.
To correlate gyrA mutations found on the Genotype MTBDRsl assay in Mycobacterium tuberculosis (MTB) isolates with Minimum Inhibitory Concentrations (MICs) to the fluoroquinolones compounds ofloxacin (OFX) and moxifloxacin (MXF).MICs for OFX and MXF were ascertained for 93 archived clinical MTB isolates that showed gyrA mutations at Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly and His. Thirty fluoroquinolones susceptible isolates as determined by presence of all wild-type gyrA bands on the Genotype MTBDRsl assay were also included.gyrA mutations at Ala90Val (n = 25), Ser91Pro (n = 6), Asp94Ala (n = 4), Asp94Asn/Tyr (n = 13), Asp94Gly (n = 42) and Asp94His (n = 3) were observed. Isolates with mutations at Ala90Val or Ser91Pro had MIC90 of 4.0 μg/ml and 1.0 μg/ml for OFX and MXF, respectively, and isolates with mutations at Asp 94Ala, Asn/Tyr, Gly and His had MIC90 of 8.0 μg/ml, and 2.5 μg/ml for OFX and MXF, respectively.MTB MICs were found to be consistently lower for MXF than for OFX among isolates with the same gyrA mutation (e.g. Ala90Val). The majority of MTB isolates containing mutations at Asp94Ala, Asn/Tyr, Gly and His in gyrA were associated with a moderate level of resistance to MXF (MIC = 2.5 μg/ml), although 3 isolates with the mutations Asp94Asn/Tyr/Gly were associated with a high level of resistance to both fluoroquinolones (MXF MICs = 5.0-8.0 μg/ml, OFX MICs = ≥10.0 μg/ml).
- Structure based virtual screening to identify inhibitors against MurE Enzyme of Mycobacterium tuberculosis using AutoDock Vina. [Journal Article]
- Bioinformation 2014; 10(11):697-702.
The Mur E enzyme of Mur pathway of Mycobacterium tuberculosis is an attractive drug target as it is unique to bacteria and is absent in mammalian cells. The virtual screening of large libraries of drug like molecules against a protein target is a common strategy used to identify novel inhibitors. However, the method has a large number of pitfalls, with large variations in accuracy caused in part by inaccurate protocols, use of improper standards and libraries, and system dependencies such as the potential for nonspecific docking from large active-site cavities. The screening of drug-like small molecules from diversity sets can, however, be used to short-list potential fragments as building blocks to generate leads with improved specificity. We describe a protocol to implement this strategy, which involves an analysis of the active site and known inhibitors to identify orthospecific determinants, virtual screening of a drug-like diversity library to identify potential drug primitives, and inspection of the potential docked fragments for both binding potential and toxicity. The protocol is implemented on the M.tb Mur E protein which has a large active site with poor enrichment of known positives and a set of drug-like molecules that meets this criteria is presented for further analysis.MTB - Mycobacterium tuberculosis, NCI - National Cancer Institute, PDB - Protein Databank.
- From workstations to workbenches: Towards predicting physicochemically viable protein-protein interactions across a host and a pathogen. [JOURNAL ARTICLE]
- IUBMB Life 2014 Dec 15.
The understanding of protein-protein interactions is indispensable in comprehending most of the biological processes in a cell. Small-scale experiments as well as large-scale high-throughput techniques over the past few decades have facilitated identification and analysis of protein-protein interactions which form the basis of much of our knowledge on functional and regulatory aspects of proteins. However, such rich catalog of interaction data should be used with caution when establishing protein-protein interactions in silico, as the high-throughput datasets are prone to false positives. Numerous computational means developed to pursue genome-wide studies on protein-protein interactions at times overlook the mechanistic and molecular details, thus questioning the reliability of predicted protein-protein interactions. We review the development, advantages, and shortcomings of varied approaches and demonstrate that by providing a structural viewpoint in terms of shape complementarity and interaction energies at protein-protein interfaces coupled with information on expression and localization of proteins homologous to an interacting pair, it is possible to assess the credibility of predicted interactions in biological context. With a focus on human pathogen Mycobacterium tuberculosis H37Rv, we show that such scrupulous use of details at the molecular level can predict physicochemically viable protein-protein interactions across host and pathogen. Such predicted interactions have the potential to provide molecular basis of probable mechanisms of pathogenesis and hence open up ways to explore their usefulness as targets in the light of drug discovery. © 2014 IUBMB Life, 2014.
- Case Report Tuberculoma masked by glioma: a case report. [Journal Article]
- Genet Mol Res 2014; 13(4):10450-3.
Tuberculous meningitis (TM), a common infectious disease of the central nervous system that is also seen in other types of tuberculosis infections, has higher mortality rates in young and middle-aged patients. TM is difficult to diagnose and treat owing to its non-specific clinical features and often atypical cerebrospinal fluid changes. Patients who present with focal neurologic signs, cough, low-grade fever and illness duration of more than 5 days, have intracalvarial abnormalities, and do not meet Thwaites' criterion findings should be diagnosed using computed tomography or magnetic resonance imaging. Mycobacterium infections can also be diagnosed by acid-fast staining of smears, cerebrospinal fluid culture, diagnostic polymerase chain reaction for Mycobacterium tuberculosis, and purified protein derivative test. To prevent TM misdiagnosis, clinicians must have sufficient knowledge of the clinical manifestations of tuberculosis. Appropriate application of tuberculosis chemotherapy drug principles, including early diagnosis and treatment, combination therapies, and consistent administration of treatment at appropriate dosages, can greatly reduce TM mortality rates and improve satisfactory treatment outcomes.
- Multi-drug resistant tuberculosis among category I treatment failures--a retrospective study. [Journal Article]
- Indian J Tuberc 2014 Apr; 61(2):148-51.
Tuberculosis (TB) remains a major global health problem and ranks as the second leading cause of death worldwide. An important cause of TB epidemic is the emergence of multi drug resistant (MDR) strains of Mycobacterium tuberculosis. Despite the availability of treatment that is expected to cure most cases of TB, levels of MDR-TB remain worryingly high in India.This study was carried out to ascertain the prevalence of MDR-TB among category I pulmonary TB treatment failure patients.This was a retrospective study involving 750 pulmonary tuberculosis patients enrolled at six district centres of Delhi State under RNTCP who failed to respond to CAT I treatment and whose sputum samples were submitted for culture and drug sensitivity testing (DST) over a period of three years (2009-2012). MDR-TB was defined as TB caused by bacilli showing resistance to at least isoniazid and rifampicin.Out of the total 750 patients included in the study, 470 (62.6 %) were culture positive. Of these, 377 (80.2%) were subjected to DST and rest 93 (19.7%) were excluded. Ultimately, DST result was available for 353 (93.6 %) cases. 239 (68%) cases were detected as multi drug resistant TB.High proportion of MDR-TB (68%) among culture positive CAT I treatment failure cases highlights the need for rapid diagnostic tests which will enable the detection of MDR-TB at an early stage and will thus minimize the risk of transmission as well as the possible errors associated with the treatment.
- Novel mutation detection IN rpoB OF rifampicin-resistant Mycobacterium Tuberculosis using pyroseouencing. [Journal Article, Research Support, Non-U.S. Gov't]
- Southeast Asian J Trop Med Public Health 2014 Jul; 45(4):843-52.
Tuberculosis (TB) remains a major global public health problem particularly severe in parts of Asia and Africa, where often it is present in HIV-AIDS patients. Although rifampicin-resistant (RIFr) TB is slow to emerge due to the low rate of mutation of its target leading to RIFE being a marker of TB that is already resistant to other anti-TB drugs, and such cases are prone to treatment failure. More than 95% of rifampicin resistance is associated with mutations in Mycobacterium tuberculosis (MTB) rpoB, with 97% of mutations occurring within the 81 bp rifampicin-resistant determining region (RRDR) of this gene. In this study, we employed pyrosequencing technique to identify mutations in RRDR and 5 codons beyond of 39 MTB strains, comprising of 14 multi-drug resistance TB (MDRTB) and 3 RIF susceptible (RIFs) MTB from the Center of Disease Control (CDC), Ratchaburi Province, and 19 mono RIFr MTB, 1 MDRTB and 2 poly-drug resistant MTB from the Chest Institute, Ministry of Public Health, Thailand. Mu- tations in 8/22 samples from the Chest Institute and 13/14 from CDC were able to be identified. Six point mutations were detected, with Ser531Leu mutation accounting for 13, the silent mutation at Gly536 for 4, deletion of Gly523 for 2, combination of His526Cys and novel Leu533Arg for 1, and a novel Leu538Arg for 1. Mutation analysis of the 81 bp fragment and 5 codons beyond in MTB rpoB using pyrosequencing provides a useful approach in predicting RIFr phenotype allowing early diagnosis and appropriate drug therapy.