scholarly journals Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

2011 ◽  
Vol 55 (5) ◽  
pp. 2032-2041 ◽  
Author(s):  
Patricia J. Campbell ◽  
Glenn P. Morlock ◽  
R. David Sikes ◽  
Tracy L. Dalton ◽  
Beverly Metchock ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
First Line ◽  
Content Type ◽  
Line Drug

ABSTRACTThe emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced:rpoB(for resistance to RIF),katGandinhA(INH),pncA(PZA),embB(EMB),gyrA(CIP and OFX), andrrs,eis, andtlyA(KAN, AMK, and CAP). A total of 314 clinicalMycobacterium tuberculosiscomplex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% forrpoB, 85.4% and 100% forkatG, 16.5% and 100% forinhA, 90.6% and 100% forkatGandinhAtogether, 84.6% and 85.8% forpncA, and 78.6% and 93.1% forembB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in theM. tuberculosiscomplex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

scholarly journals Evaluation of the GenoType MTBDR sl Version 2.0 Assay for Second-Line Drug Resistance Detection of Mycobacterium tuberculosis Isolates in South Africa

2016 ◽  
Vol 55 (3) ◽  
pp. 791-800 ◽  
Author(s):  
Y. Gardee ◽  
A. W. Dreyer ◽  
H. J. Koornhof ◽  
S. V. Omar ◽  
P. da Silva ◽  
...  
Keyword(s):  
South Africa ◽  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Sensitivity And Specificity ◽  
Drug Susceptibility Testing ◽  
Rapid Screening ◽  
Second Line ◽  
Content Type ◽  
Version 2.0 ◽  
Line Drug

ABSTRACT Early detection of resistance to second-line antituberculosis drugs is important for the management of multidrug-resistant tuberculosis (MDR-TB). The GenoType MTBDR sl version 2.0 (VER 2.0) line probe assay has been redesigned for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) ( gyrA and gyrB genes) and second-line injectable drugs (SLID) ( rrs and eis genes). The study evaluated the diagnostic performance of the GenoType MTBDR sl VER 2.0 assay for the detection of second-line drug resistance compared with phenotypic drug susceptibility testing (DST), using the Bactec MGIT 960 system on Mycobacterium tuberculosis complex isolates from South Africa. A total of 268 repository isolates collected between 2012 and 2014, which were rifampin monoresistant or MDR based on DST, were selected. MTBDR sl VER 2.0 testing was performed on these isolates and the results analyzed. The MTBDR sl VER 2.0 sensitivity and specificity indices for culture isolates were the following: FLQ, 100% (95% confidence interval [CI] 95.8 to 100%) and 98.9% (95% CI, 96.1 to 99.9%); SLID, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.7 to 99.7%). The sensitivity and specificity observed for individual SLID were the following: amikacin, 93.8% (95% CI, 79.2 to 99.2%) and 98.5% (95% CI, 95.5 to 99.7%); kanamycin, 89.2% (95% CI, 79.1 to 95.6%) and 98.5% (95% CI, 95.5 to 99.7%); and capreomycin, 86.2% (95% CI, 68.3 to 96.1%) and 95.9% (95% CI, 92.2 to 98.2%). An interoperator reproducibility of 100% and an overall interlaboratory performance of 93% to 96% were found. The overall improvement in sensitivity and specificity with excellent reproducibility makes the GenoType MTBDR sl VER 2.0 a highly suitable tool for rapid screening of clinical isolates for second-line drug resistance for use in high-burden TB/HIV settings.

scholarly journals Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

2013 ◽  
Vol 58 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Jongseok Lee ◽  
Derek T. Armstrong ◽  
Willy Ssengooba ◽  
Jeong-ae Park ◽  
Yeuni Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Critical Concentration ◽  
Microtiter Plate ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
Content Type ◽  
The Republic ◽  
Phenotypic Methods

ABSTRACTForMycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB forM. tuberculosisdrug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archivedM. tuberculosisisolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results forM. tuberculosissusceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

scholarly journals A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid

2016 ◽  
Vol 54 (12) ◽  
pp. 2963-2968 ◽  
Author(s):  
Koné Kaniga ◽  
Daniela M. Cirillo ◽  
Sven Hoffner ◽  
Nazir A. Ismail ◽  
Devinder Kaur ◽  
...  
Keyword(s):  
Quality Control ◽  
Reference Strain ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
Broth Microdilution ◽  
First Line ◽  
Antituberculosis Drugs ◽  
Content Type

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain wasMycobacterium tuberculosisH37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 μg/ml), rifampin (0.03 to 0.25 μg/ml), ethambutol (0.25 to 2 μg/ml), levofloxacin (0.12 to 1 μg/ml), moxifloxacin (0.06 to 0.5 μg/ml), ofloxacin (0.25 to 2 μg/ml), amikacin (0.25 to 2 μg/ml), kanamycin (0.25 to 2 μg/ml), capreomycin (0.5 to 4 μg/ml), linezolid (0.25 to 2 μg/ml), and clofazimine (0.03 to 0.25 μg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against theM. tuberculosisH37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

scholarly journals Performance of the New Version (v2.0) of the GenoType MTBDRslTest for Detection of Resistance to Second-Line Drugs in Multidrug-Resistant Mycobacterium tuberculosis Complex Strains

2016 ◽  
Vol 54 (6) ◽  
pp. 1573-1580 ◽  
Author(s):  
Florence Brossier ◽  
David Guindo ◽  
Anne Pham ◽  
Florence Reibel ◽  
Wladimir Sougakoff ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Drug Susceptibility Testing ◽  
Second Line ◽  
Content Type ◽  
Injectable Drugs ◽  
Resistant Tuberculosis

Detecting resistance to fluoroquinolones (FQ) and second-line injectable drugs (amikacin [AMK], kanamycin [KAN], and capreomycin [CAP]) is crucial given the worldwide increase in the incidence of extensively drug-resistant tuberculosis (XDR-TB). A new version of the GenoType MTBDRsltest (v2.0) has been developed to improve the detection of resistance to FQ (involvinggyrAandgyrBmutations) and to second-line injectable drugs (involvingrrsandeispromoter mutations) inMycobacterium tuberculosis. A collection of 127 multidrug-resistant (MDR)M. tuberculosiscomplex strains was tested using the first (v1) and second (v2.0) versions of the MTBDRsltest, as well as DNA sequencing. The specificities in resistance detection of v1 and v2.0 were similar throughout, whereas the levels of sensitivity of v2.0 were superior for FQ (94.8% versus 89.6%) and KAN (90.5% versus 59.5%) but similar for AMK (91.3%) and CAP (83.0%). The sensitivity and specificity of v2.0 were superior to those of v1 for the detection of pre-XDR strains (83.3% versus 75.0% and 88.6% versus 67.1%, respectively), whereas the sensitivity of v2.0 was superior to that of v1 only for the detection of XDR strains (83.0% versus 49.1%). In conclusion, MTBDRslv2.0 is superior to MTBDRslv1 and efficiently detects the most common mutations involved in resistance to FQ and aminoglycosides/CAP. However, due to mutations not recognized by v2.0 or to the presence of resistance mechanisms not yet characterized (particularly mechanisms related to monoresistance to aminoglycosides or CAP), the results for wild-type strains obtained with MTBDRslv2.0 should be confirmed by further DNA sequencing and phenotypic drug susceptibility testing.

scholarly journals Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

2016 ◽  
Vol 54 (8) ◽  
pp. 2058-2067 ◽  
Author(s):  
Rebecca E. Colman ◽  
Julia Anderson ◽  
Darrin Lemmer ◽  
Erik Lehmkuhl ◽  
Sophia B. Georghiou ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Amplicon Sequencing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Health Concern ◽  
Clinical Samples ◽  
Drug Resistant ◽  
Content Type

Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing ofMycobacterium tuberculosisDNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.

scholarly journals Comparison of the Performances of Two In-House Rapid Methods for Antitubercular Drug Susceptibility Testing

2008 ◽  
Vol 53 (2) ◽  
pp. 808-810 ◽  
Author(s):  
Agustina I. de la Iglesia ◽  
Emma J. Stella ◽  
Héctor R. Morbidoni
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Sensitivity And Specificity ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Antitubercular Drug ◽  
Rapid Methods ◽  
Resistance Assessment

ABSTRACT Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

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