Quantitative Analysis of mRNA as a Marker for Viability  of Mycobacterium tuberculosis

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.

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Detection of Viable Mycobacterium tuberculosis by Reverse Transcriptase-Strand Displacement Amplification of mRNA

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.518-523.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 518-523 ◽

Cited By ~ 46

Author(s):

T. J. Hellyer ◽

L. E. DesJardin ◽

L. Teixeira ◽

M. D. Perkins ◽

M. D. Cave ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Reverse Transcriptase ◽

Therapeutic Efficacy ◽

Sputum Sample ◽

Drug Susceptibility ◽

Strand Displacement ◽

Strand Displacement Amplification ◽

Amplification System

Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability. The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of M. tuberculosis α-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis. The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively. In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for α-antigen DNA. The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log10 CFU/ml/day. Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.

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Molecular Analysis of Codon 548 in therpoBGene Involved in Mycobacterium tuberculosis Resistance to Rifampin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04374-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1542-1548 ◽

Cited By ~ 7

Author(s):

Yu-Tze Horng ◽

Wen-Yih Jeng ◽

Yih-Yuan Chen ◽

Che-Hung Liu ◽

Horng-Yunn Dou ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Resistance ◽

Drug Susceptibility ◽

Content Type ◽

Stable Hydrogen Bond ◽

Resistance Patterns ◽

Resistant Strains ◽

Rifampin Resistance ◽

Susceptibility Tests ◽

Dna Complex

ABSTRACTMostMycobacterium tuberculosisrifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the generpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinantMycobacterium smegmatisandM. tuberculosisisolates carrying mutatedrpoB(Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of therpoBgene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

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Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of pncA PCR Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.00145-19 ◽

2019 ◽

Vol 57 (8) ◽

Author(s):

Kingsley King-Gee Tam ◽

Kenneth Siu-Sing Leung ◽

Gilman Kit-Hang Siu ◽

Kwok-Chiu Chang ◽

Samson Sai-Yin Wong ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Direct Detection ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Activity Data ◽

Content Type ◽

Treatment Regimens ◽

Mdr Tb ◽

Resistant Strains ◽

Respiratory Specimens

ABSTRACT An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtime MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

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Microscopic Observation Drug Susceptibility (MODS) Assay: A Convenient Method for Determining Antibiogram of Clinical Isolates of Mycobacterium tuberculosis in Ghana

Medical Sciences ◽

10.3390/medsci8010005 ◽

2020 ◽

Vol 8 (1) ◽

pp. 5

Author(s):

Enid Owusu ◽

Mercy Jemima Newman

Keyword(s):

Mycobacterium Tuberculosis ◽

Rural Areas ◽

Microscopic Observation ◽

Drug Susceptibility ◽

Ease Of Use ◽

Health Centers ◽

Culture Methods ◽

Susceptibility Tests ◽

User Friendly

(1) Background: Present methods for drug susceptibility tests (DST) rely on culture methods that are sophisticated and relatively faster, or a slow and cheaper option. These methods frustrate disease control; therefore, there is a need for methods that incorporate key functions of microscopy and culture, with reduced cost burden and sophistry. Thus, the purpose of this study was to identify which, among the most commonly used (in Ghana) methods, can conveniently be used at health centers located in rural areas for effective DST determination of Mycobacterium tuberculosis (MTB). (2) Methods: Mycobacterium tuberculosis isolates were tested for their susceptibility to streptomycin, isoniazid, rifampicin, ethambutol (SIRE), and pyrazinamide by microscopic observation drug susceptibility (MODS) and BACTEC MGIT 960 methods. Evaluations were based on shorter turnaround periods, rapidity, ease of use, cost, etc. A comparative analysis was statistically expressed as kappa values. (3) Results: Endpoints for drug susceptibilities by MODS averaged 13 days (7–32), whilst that for BACTEC MGIT 960 was 10 days with a further 12 days to detect resistance. Therefore, a turnaround period of 22 days was needed for DST by BACTEC MGIT 960, compared to 13 days for MODS. There were differences in correlation levels between the two methods, as determined by their kappa values. (4) Conclusion: The MODS assay was found to be less costly, more user-friendly, and still able to be conveniently used at health centers located in rural areas known to be endemic for TB, particularly in Ghana.

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Determination of 16S rRNA Sequences of Streptococcus mitis and Streptococcus gordonii and Phylogenetic Relationships among Members of the Genus Streptococcus

International Journal of Systematic Bacteriology ◽

10.1099/00207713-45-2-406 ◽

1995 ◽

Vol 45 (2) ◽

pp. 406-408 ◽

Cited By ~ 287

Author(s):

Y. KAWAMURA ◽

X.-G. HOU ◽

F. SULTANA ◽

H. MIURA ◽

T. EZAKI

Keyword(s):

16S Rrna ◽

Phylogenetic Relationships ◽

Streptococcus Gordonii ◽

Streptococcus Mitis ◽

Rrna Sequences ◽

16S Rrna Sequences

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Acetylation of isoniazid - a novel mechanism of isoniazid resistance in Mycobacterium tuberculosis

10.1101/2020.02.10.941252 ◽

2020 ◽

Cited By ~ 1

Author(s):

K. B. Arun ◽

Aravind Madhavan ◽

Billu Abraham ◽

M. Balaji ◽

K. C. Sivakumar ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Isonicotinic Acid ◽

Active Form ◽

Drug Susceptibility ◽

Cell Wall Biosynthesis ◽

First Line ◽

Isoniazid Resistance ◽

Acetyl Group ◽

Resistant Strains ◽

Susceptibility Assay

AbstractIsoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a pro-drug which is converted into its active form by the intracellular KatG enzyme of Mycobacterium tuberculosis. The activated drug hinders cell wall biosynthesis by inhibiting InhA protein. INH resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze transfer of acetyl group to INH from acetyl CoA. HPLC and LC-MS analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. Drug susceptibility assay and confocal analysis showed that M. smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at minimum inhibitory concentrations that inhibited wildtype strains. In addition, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity.

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A Simple System for the Rapid Determination of the Anaerobic Compensation Point of Plant Tissue

HortScience ◽

10.21273/hortsci.25.4.480 ◽

1990 ◽

Vol 25 (4) ◽

pp. 480-482 ◽

Cited By ~ 4

Author(s):

Jeffrey A. Leshuk ◽

Mikal E. Saltveit

Keyword(s):

Plant Tissue ◽

Rapid Determination ◽

Compensation Point ◽

Simple System ◽

Traditional Methods ◽

Time Required

A method is described for the rapid determination of the anaerobic compensation point (ACP) of plant tissue, i.e., the O2 concentration at which CO2 production is minimum. The rate of CO2 production is measured from tissue exposed to an exponentially declining O2 concentration produced by a flow of N2 into a dilution bottle initially containing air. Too rapid a rate of O2 decline produces abnormal data because of the time required for the tissue to respond to changes in O2 concentration. The ACP is easily determined from a plot of CO2 production vs. O2 concentration. Rates of CO2 production and ACPS calculated using the exponentially declining system are similar to those calculated from traditional methods of continuously holding tissue under various O2 concentrations.

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A Comprehensive Evaluation of GeneLEAD VIII DNA Platform Combined to Deeplex Myc-TB® Assay to Detect in 8 Days Drug Resistance to 13 Antituberculous Drugs and Transmission of Mycobacterium tuberculosis Complex Directly From Clinical Samples

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.707244 ◽

2021 ◽

Vol 11 ◽

Author(s):

Isabelle Bonnet ◽

Vincent Enouf ◽

Florence Morel ◽

Vichita Ok ◽

Jérémy Jaffré ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Comprehensive Evaluation ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Clinical Samples ◽

Routine Practice ◽

Single Nucleotide Variants ◽

Sequencing Platform

The GeneLEAD VIII (Diagenode, Belgium) is a new, fully automated, sample-to-result precision instrument for the extraction of DNA and PCR detection of Mycobacterium tuberculosis complex (MTBC) directly from clinical samples. The Deeplex Myc-TB® assay (Genoscreen, France) is a diagnostic kit based on the deep sequencing of a 24-plexed amplicon mix allowing simultaneously the detection of resistance to 13 antituberculous (antiTB) drugs and the determination of spoligotype. We evaluated the performance of a strategy combining the both mentioned tools to detect directly from clinical samples, in 8 days, MTBC and its resistance to 13 antiTB drugs, and identify potential transmission of strains from patient-to-patient. Using this approach, we screened 112 clinical samples (65 smear-negative) and 94 MTBC cultured strains. The sensitivity and the specificity of the GeneLEAD/Deeplex Myc-TB approach for MTBC detection were 79.3% and 100%, respectively. One hundred forty successful Deeplex Myc-TB results were obtained for 46 clinical samples and 94 strains, a total of 85.4% of which had a Deeplex Myc-TB susceptibility and resistance prediction consistent with phenotypic drug susceptibility testing (DST). Importantly, the Deeplex Myc-TB assay was able to detect 100% of the multidrug-resistant (MDR) MTBC tested. The lowest concordance rates were for pyrazinamide, ethambutol, streptomycin, and ethionamide (84.5%, 81.5%, 73%, and 55%, respectively) for which the determination of susceptibility or resistance is generally difficult with current tools. One of the main difficulties of Deeplex Myc-TB is to interpret the non-synonymous uncharacterized variants that can represent up to 30% of the detected single nucleotide variants. We observed a good level of concordance between Deeplex Myc-TB-spoligotyping and MIRU-VNTR despite a lower discriminatory power for spoligotyping. The median time to obtain complete results from clinical samples was 8 days (IQR 7–13) provided a high-throughput NGS sequencing platform was available. Our results highlight that the GeneLEAD/Deeplex Myc-TB approach could be a breakthrough in rapid diagnosis of MDR TB in routine practice.

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Multivariate Determination of Sugar Powders by Attenuated Total Reflectance Infrared Spectroscopy

Applied Spectroscopy ◽

10.1366/0003702934334949 ◽

1993 ◽

Vol 47 (4) ◽

pp. 452-457 ◽

Cited By ~ 11

Author(s):

N. Dupuy ◽

M. Meurens ◽

B. Sombret ◽

P. Legrand ◽

J.P. Huvenne

Keyword(s):

Quantitative Analysis ◽

Attenuated Total Reflectance ◽

Infrared Spectrometry ◽

Infrared Analysis ◽

Total Reflectance ◽

Regression Methods ◽

Peak Resolution ◽

Atr Spectroscopy ◽

Time Required

Thanks to what has been achieved by Fourier transformation, infrared analysis can now become a state-of-the-art device in quality control laboratories if we consider its precision and the gain in time it ensures. Moreover, an increasing number of new mathematical regression methods such as partial least-squares (PLS) regression allow multicomponent quantitative analysis in mixtures. Nevertheless, the efficiency of infrared spectrometry as a quantitative analytical method often depends on the choice of an adequate presentation for the sample. For quantitative analysis of powders, sampling appears difficult in the mid-IR. We have developed a method built on three advantages: the rapidity of Fourier transform spectroscopy, fast solvent elimination, and good peak resolution. For enhancing peak intensity on attenuated total reflectance (ATR) spectroscopy we use nondissolving and evaporating liquids. For instance, the analysis of three components (glucose, fructose, and sucrose) can be done with a precision in the range of 4%, whereas the time required to obtain an analysis report is about 5 min.

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