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

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.

scholarly journals Rapid, Efficient Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Sputum by Microscopic Observation of Broth Cultures

2000 ◽  
Vol 38 (3) ◽  
pp. 1203-1208 ◽  
Author(s):  
Luz Caviedes ◽  
Tien-Shun Lee ◽  
Robert H. Gilman ◽  
Patricia Sheen ◽  
Emily Spellman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Microscopic Observation ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Multiple Drug ◽  
Specific Method ◽  
Culture Methods ◽  
Efficient Detection ◽  
Multiple Drug Resistant

Inexpensive, rapid, and reliable methods of detecting infection by and drug susceptibility of Mycobacterium tuberculosis (MTB) are crucial to the control of tuberculosis. The novel microscopic observation broth-drug susceptibility assay (MODS) detects early growth of MTB in liquid medium, allowing more timely diagnosis and drug susceptibility testing. Sputum samples from hospitalized patients in Peru were analyzed by using stains, culture, and PCR. Sensitivity of MODS (92%) compared favorably with the most sensitive of the other culture methods (93%). Sputum samples positive for tuberculosis were tested for susceptibility to isoniazid and rifampin with the microwell alamar blue assay (MABA) and MODS. In 89% of cases, there was concordance between MODS and MABA. Of the diagnostic and susceptibility testing methods used, MODS yielded results most rapidly (median, 9.0 and 9.5 days, respectively). MODS is a rapid, inexpensive, sensitive, and specific method for MTB detection and susceptibility testing; it is particularly appropriate for use in developing countries burdened by significant infection rates and increasing numbers of multiple-drug-resistant cases.

scholarly journals Black Cumin (Nigella sativa) Against Mycobacterium tuberculosis Strain H37RV And MDR-TB

Elkawnie ◽  
2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Mashuri Masri ◽  
Cut Muthiadin ◽  
Masita Masita ◽  
Tri Cahyanto ◽  
Lianah Lianah ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistance ◽  
Microscopic Observation ◽  
Nigella Sativa ◽  
Drug Susceptibility ◽  
Tuberculosis Strain ◽  
Black Cumin ◽  
Mdr Tb ◽  
Susceptibility Assay ◽  
Strain H37rv

Abstract: Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis. 10 million people suffer from TB Every year. Although TB is a preventable and treatable disease, 1.5 million people die every year due to TB. Alternative treatments continue to be pursued, and treatment with the latest TB drugs that are continuously being encouraged. Black cumin (Nigella sativa) seed contains essential oils with active compounds such as thymohydroquinone, Oleoresins, flavonoids, alkaloids, saponins, tannins, and terpenoids that act as antibacterial drugs. This study aims to determine the sensitivity of  N. sativa seed extract in inhibiting the growth of  M. tuberculosis strain H37RV and MDR-TB (Multidrug Resistance-TB). This research using Microscopic-Observation and Drug-Susceptibility Assay (MODS) method. Extraction of N. sativa was carried out by the maceration method using 70% methanol as a solvent. The results showed that the M. tuberculosis strain H37RV and MDR-TB were sensitive to N. sativa extract at concentrations of 5 and 10% but resistant to N. sativa extract at concentrations of 1 and 3%.Abstrak: Tuberkulosis (TB) adalah penyakit menular yang disebabkan oleh Bakteri Mycobacterium tuberculosis. Penyakit ini menimbulkan dampak kematian yang cukup mengkhawatirkan.  Penyakit tersebut dapat dicegah dan diobati. Salah satu sumber pengobatannya menggunakan biji jintan hitam (Nigella sativa) yang mengandung minyak atsiri dengan senyawa aktif seperti timohidrokuinon, oleoresin, flavonoid, alkaloid, saponin, tanin, dan terpenoid yang berfungsi sebagai obat antibakteri. Penelitian ini bertujuan untuk mengetahui sensitivitas ekstrak biji N. sativa dalam menghambat pertumbuhan M. tuberculosis strain H37RV and MDR-TB (Multidrug-Resistance-TB). Penelitian ini menggunakan metode Microscopic-Observation and Drug-Susceptibility Assay (MODS). Ekstraksi N. sativa dilakukan dengan metode maserasi menggunakan pelarut metanol 70%. Hasil yang diperoleh menunjukkan bahwa bakteri M. tuberculosis strain H37RV dan TB-MDR, kedua  strain tsb sensitif terhadap ekstrak N. sativa konsentrasi 5 dan 10%,  tetapi resisten terhadap  ekstrak N. sativa konsentrasi 1 dan 3%.

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

2014 ◽  
Vol 59 (3) ◽  
pp. 1542-1548 ◽  
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.

scholarly journals Trends in Testing for Mycobacterium tuberculosis Complex From US Public Health Laboratories, 2009–2013

2016 ◽  
Vol 132 (1) ◽  
pp. 56-64 ◽  
Author(s):  
Frances Tyrrell ◽  
Cortney Stafford ◽  
Mitchell Yakrus ◽  
Monica Youngblood ◽  
Andrew Hill ◽  
...  
Keyword(s):  
Public Health ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
The United States ◽  
Drug Susceptibility ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Tuberculosis Complex ◽  
Complex Detection ◽  
Susceptibility Tests

Objective: We investigated data from US public health laboratories funded through the Centers for Disease Control and Prevention’s Tuberculosis Elimination and Laboratory Cooperative Agreement to document trends and challenges in meeting national objectives in tuberculosis (TB) laboratory diagnoses. Methods: We examined data on workload and turnaround time from public health laboratories’ progress reports during 2009-2013. We reviewed methodologies, laboratory roles, and progress toward rapid detection of Mycobacterium tuberculosis complex through nucleic acid amplification (NAA) testing. We compared selected data with TB surveillance reports to estimate public health laboratories’ contribution to national diagnostic services. Results: During the study period, culture and drug susceptibility tests decreased, but NAA testing increased. Public health laboratories achieved turnaround time benchmarks for drug susceptibility tests at lower levels than for acid-fast bacilli smear and identification from culture. NAA positivity in laboratories among surveillance-reported culture-positive TB cases increased from 26.6% (2355 of 8876) in 2009 to 40.0% (2948 of 7358) in 2013. Public health laboratories provided an estimated 50.9% (4285 of 8413 in 2010) to 57.2% (4210 of 7358 in 2013) of culture testing and 88.3% (6822 of 7727 in 2011) to 94.4% (6845 of 7250 in 2012) of drug susceptibility tests for all US TB cases. Conclusions: Public health laboratories contribute substantially to TB diagnoses in the United States. Although testing volumes mostly decreased, the increase in NAA testing indicates continued progress in rapid M tuberculosis complex detection.

Proficiency testing of conventional drug susceptibility tests of Mycobacterium tuberculosis

1987 ◽  
Vol 33 (12) ◽  
pp. 1064-1068 ◽  
Author(s):  
Adalbert Laszlo ◽  
Dorothy M. Helbecque ◽  
Walter Tostowaryk
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proficiency Testing ◽  
Susceptibility Testing ◽  
Developed Countries ◽  
Drug Susceptibility ◽  
Reference Laboratory ◽  
In Vitro Susceptibility ◽  
Testing Procedures ◽  
Drug Concentrations ◽  
Susceptibility Tests

Proficiency testing of indirect drug susceptibility tests of Mycobacterium tuberculosis was begun in 1985 by the Laboratory Centre for Disease Control (LCDC) with the participation of Provincial Public Health Laboratories in Canada. Comparable sets of 60 cultures of Mycobacterium tuberculosis representing 30 strains were distributed by LCDC to the participating laboratories to be tested for drug susceptibility against isoniazid, streptomycin, rifampin, and ethambutol using conventional methodologies. Intralaboratory agreement values determined by comparing results obtained on sets of duplicate cultures were high and were found to vary little from drug to drug and from laboratory to laboratory. Interlaboratory agreement was determined by comparing results reported by participating laboratories to those obtained by the Reference Laboratory. Agreement percentages were found to be lower for drug-resistant cultures than for drug-susceptible cultures. The reliability of drug susceptibility testing results was higher for isoniazid and rifampin, than for ethambutol and streptomycin. This study shows that the higher subsidiary drug concentrations do not compare well with main drug concentrations, especially in the case of streptomycin and ethambutol. The significance of the higher subsidiary concentrations in in vitro susceptibility testing is therefore in need of clarification. The proficiency testing results obtained in this study compare favorably with those reported in other developed countries despite the fact that a variety of testing procedures are used throughout the country.

Improved Recovery of Mycobacterium tuberculosis From Children Using the Microscopic Observation Drug Susceptibility Method

PEDIATRICS ◽  
2006 ◽  
Vol 118 (1) ◽  
pp. e100-e106 ◽  
Author(s):  
R. A. Oberhelman ◽  
G. Soto-Castellares ◽  
L. Caviedes ◽  
M. E. Castillo ◽  
P. Kissinger ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Microscopic Observation ◽  
Drug Susceptibility

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

1999 ◽  
Vol 37 (2) ◽  
pp. 290-295 ◽  
Author(s):  
T. J. Hellyer ◽  
L. E. DesJardin ◽  
G. L. Hehman ◽  
M. D. Cave ◽  
K. D. Eisenach
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Quantitative Analysis ◽  
16S Rrna ◽  
Therapeutic Efficacy ◽  
Rapid Determination ◽  
Drug Susceptibility ◽  
Resistant Strains ◽  
Time Required ◽  
Rrna Sequences

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.

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

1999 ◽  
Vol 37 (3) ◽  
pp. 518-523 ◽  
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.

scholarly journals Comparison between the conventional method and molecular line probe assay for identification and drug sensitivity of mycobacteria tuberculosis from clinical specimens

2014 ◽  
Vol 2 (03) ◽  
pp. 80-83
Author(s):  
Ritu Kansal ◽  
Molly Madan ◽  
Richa Kansal ◽  
Vivek Agwan ◽  
Isha Bansal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conventional Method ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Line Probe Assay ◽  
Molecular Method ◽  
Traditional Drug ◽  
Probe Assay ◽  
Susceptibility Tests ◽  
Mycobacteria Tuberculosis

Rapid susceptibility testing of Mycobacterium tuberculosis strains is imperative for therapy selection but traditional drug susceptibility tests take weeks or are expensive. Classical drug susceptibility (DST) may take up to 2 to 4 months. The line probe assay is a commercially available line-probe assay that rapidly detects Mycobacterium tuberculosis (MTB) complex, as well as the most common mutations associated with rifampicin and isoniazid. In this study we assessed the sensitivity and specificity of the rapid molecular method in comparison with the conventional method.

scholarly journals 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

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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