scholarly journals Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification.

1997 ◽  
Vol 35 (5) ◽  
pp. 1239-1243 ◽  
Author(s):  
F Z Badak ◽  
D L Kiska ◽  
M O'Connell ◽  
C M Nycz ◽  
C Hartley ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Mycobacterial Growth ◽  
Indicator Tubes ◽  
Growth Indicator

scholarly journals Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization

1996 ◽  
Vol 42 (10) ◽  
pp. 1604-1608 ◽  
Author(s):  
G T Walker ◽  
C P Linn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Restriction Enzyme ◽  
Fluorescence Polarization ◽  
Target Sequence ◽  
Strand Displacement ◽  
Insertion Element ◽  
Strand Displacement Amplification ◽  
Simple Protocol ◽  
Polymerase Binding

Abstract Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.

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 Reduction of Contamination of Mycobacterial Growth Indicator Tubes with a Modified Antimicrobial Combination

2002 ◽  
Vol 40 (10) ◽  
pp. 3845-3847 ◽  
Author(s):  
C. L. Chang ◽  
T. S. Park ◽  
S. H. Oh ◽  
H. H. Kim ◽  
E. Y. Lee ◽  
...  
Keyword(s):  
Mycobacterial Growth ◽  
Indicator Tubes ◽  
Growth Indicator

Evaluation of the BBL MGIT (Mycobacterial Growth Indicator Tube) AST SIRE System for Antimycobacterial Susceptibility Testing of Mycobacterium tuberculosis to 4 Primary Antituberculous Drugs

2000 ◽  
Vol 124 (1) ◽  
pp. 82-86
Author(s):  
John S. Bergmann ◽  
Geoffrey Fish ◽  
Gail L. Woods
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Clinical Isolates ◽  
Indicator Tube ◽  
The Mean ◽  
Inoculum Preparation ◽  
Mycobacterial Growth ◽  
Mean Time ◽  
Mycobacterial Growth Indicator Tube ◽  
Growth Indicator

Abstract Objective.—To evaluate the performance of the BBL MGIT (Mycobacterial Growth Indicator Tube) AST SIRE system for the antimycobacterial susceptibility testing of Mycobacterium tuberculosis to isoniazid (at a concentration equivalent to the lower concentration used for testing by the method of proportion), rifampin, ethambutol, and streptomycin. Design.—Thirty-one clinical isolates and 30 challenge strains provided by the Centers for Disease Control and Prevention (CDC) were tested by MGIT AST SIRE using 2 methods of inoculum preparation, and results were compared with those of the method of proportion, which was considered the reference method. Clinical isolates for which the results of the 2 methods were discordant also were tested at 2 reference laboratories. Results.—Based on data from our site and the reference laboratories, agreement rates between initial MGIT AST SIRE results and the method of proportion for the clinical isolates with the inoculum prepared from a McFarland equivalent and from a positive MGIT tube, respectively, were 100% and 96.8% for isoniazid, 100% and 100% for rifampin, 96.8% and 100% for ethambutol, and 100% and 100% for streptomycin, excluding the isolate for which the discordant streptomycin result could not be resolved. For the 30 challenge isolates, agreement rates between MGIT AST SIRE and expected results and between method of proportion and expected results, respectively, were 96.7% and 93.3% for isoniazid, 93.3% and 100% for rifampin, 83.3% and 100% for ethambutol, and 93.3% and 100% for streptomycin. For the clinical isolates, the mean time to an MGIT AST SIRE result of susceptible was 6.15 ± 0.13 days (range, 5–8 days). For a result of resistant, the mean time overall was 5.00 ± 0.24 days (range, 3–8 days). Conclusion.—These data suggest that the MGIT AST SIRE system, using either method of inoculum preparation, is an acceptable alternative to the BACTEC 460 TB method of susceptibility testing of clinical isolates of M tuberculosis to isoniazid, rifampin, ethambutol, and streptomycin. Reasons for the lower agreement with the CDC challenge isolates should be investigated. Further evaluation of the MGIT AST SIRE system using a concentration of isoniazid equivalent to the higher concentration tested by the method of proportion would be useful, because the decision concerning use of this agent generally is based on the susceptibility test result at the higher concentration.

scholarly journals Validating a 14-Drug Microtiter Plate Containing Bedaquiline and Delamanid for Large-Scale Research Susceptibility Testing ofMycobacterium tuberculosis

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Paola M. V. Rancoita ◽  
Federica Cugnata ◽  
Ana Luíza Gibertoni Cruz ◽  
Emanuele Borroni ◽  
Sarah J. Hoosdally ◽  
...  
Keyword(s):  
Large Scale ◽  
Critical Concentration ◽  
Microtiter Plate ◽  
Content Type ◽  
Proportion Method ◽  
Resistance Prediction ◽  
Mycobacterial Growth ◽  
Interlaboratory Reproducibility ◽  
Indicator Tubes ◽  
Growth Indicator

ABSTRACTThe UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinicalMycobacterium tuberculosisisolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.

scholarly journals Comparison of MGIT 960 with Lowenstein Jensen Media for Recovery of Mycobacteria from Extrapulmonary Specimens in Southern India

2021 ◽  
Author(s):  
Kalpana Thangavelu ◽  
Imola Jamir ◽  
Kalaiarasan Ellappan ◽  
Krishnapriya Krishnakumariamma ◽  
Pallam Gopichand ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Extrapulmonary Tuberculosis ◽  
Tertiary Care ◽  
Smear Microscopy ◽  
Care Hospital ◽  
Lowenstein Jensen ◽  
Time To Detection ◽  
Culture Positive ◽  
Indicator Tubes ◽  
Growth Indicator

Introduction: Prevalence of Extrapulmonary Tuberculosis (EPTB) due to Mycobacteriumtuberculosis and Non-Tuberculous Mycobacteria (NTM) are on the rise especially in a developing country like India. Smear Microscopy (SM) is commonly used for detection of mycobacteria. Due to the paucibacillary nature in the extrapulmonary specimens SM pose a problem in detection. Though molecular methods are increasingly used now-a-days but there are possibilities that these reactions may get inhibited due to the presence of inhibitors in the extrapulmonary specimens. Aim: To compare Mycobacterium Growth Indicator Tubes (MGIT 960) with Lowenstein Jensen (LJ) medium for the detection of mycobacteria. Materials and Methods: The current prospective study was conducted on 1879 extrapulmonary specimens collected from a tertiary care hospital during the study period from July 2018 to March 2020. Specimens were subjected to Ziehl Neelsen (ZN) staining and Auramine Phenol (AP) staining. Culture was done in both LJ media and MGIT 960 culture. Positive mycobacterial cultures were subjected to MPT64 Immunochromatographic Test (ICT). Data were analysed using the Statistical Package for Social Sciences (SPSS®) for Windows® release 21.0 (SPSS Inc., Chicago, IL, USA). Results: A total of 129 (6.9%) and 105 (5.6%) mycobacteria was isolated by MGIT 960 and by LJ culture respectively among 1879 extrapulmonary specimens. MGIT 960 identified 118 (91.5%) as Mycobacterium tuberculosis complex and 11 (8.5%) as NTM among the total mycobacteria isolated. Out of 105 mycobacteria grown by LJ culture, 95 (90.5%) and 10 (9.5%) were identified as Mycobacterium tuberculosis and NTM, respectively. The rate of contamination associated with MGIT 960 and LJ culture was 4.6% and 4.3% respectively. The Time to Detection (TTD) was found to be significantly shorter for isolation of Mycobacterium tuberculosis by MGIT 960 culture compared to LJ culture. Conclusion: In the current study, authors compared MGIT 960 with solid LJ culture for recovery of both Mycobacterium tuberculosisComplex and NTM from extrapulmonary specimens and authors found increased recovery by MGIT 960 compared to LJ culture and also shorter duration of detection for Mycobacterium tuberculosis by MGIT 960 with comparable contamination rates.

scholarly journals Comparison of MGIT and Myco/F Lytic Liquid-Based Blood Culture Systems for Recovery of Mycobacterium tuberculosis from Pleural Fluid

2015 ◽  
Vol 53 (4) ◽  
pp. 1391-1394 ◽  
Author(s):  
Elizabeth Harausz ◽  
John Kafuluma Lusiba ◽  
Mary Nsereko ◽  
John L. Johnson ◽  
Zahra Toossi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pleural Fluid ◽  
Culture System ◽  
Liquid Culture ◽  
Content Type ◽  
Indicator Tube ◽  
Liquid Culture System ◽  
Mycobacterial Growth ◽  
Mycobacterial Growth Indicator Tube ◽  
Growth Indicator

The specificities and sensitivities of the Bactec mycobacterial growth indicator tube (MGIT) system for the recovery ofMycobacterium tuberculosisfrom pleural fluid are not statistically different than those of the Myco/F lytic liquid culture system. The time to positivity is shorter in the MGIT system (12.7 versus 20.7 days, respectively;P= 0.007). The Myco/F lytic culture system may be an alternative to the MGIT system for diagnosing pleural tuberculosis.

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