scholarly journals Mycobacterium tuberculosis Population Structure Determines the Outcome of Genetics-Based Second-Line Drug Resistance Testing

2012 ◽  
Vol 56 (5) ◽  
pp. 2420-2427 ◽  
Author(s):  
E. M. Streicher ◽  
I. Bergval ◽  
K. Dheda ◽  
E. C. Böttger ◽  
N. C. Gey van Pittius ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Population Structure ◽  
Drug Susceptibility ◽  
Resistance Testing ◽  
Second Line ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Resistant Tuberculosis ◽  
Emergence Of Resistance

ABSTRACTThe global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in thegyrAandgyrBgenes and therrsgene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% ofMycobacterium tuberculosisisolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in thegyrAorrrsgene. These clones harbored previously described mutations in either thegyrAorrrsgene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

scholarly journals Genotyping and Prevalence of Pyrazinamide- and Moxifloxacin-Resistant Tuberculosis in China, 2000 to 2010

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Yu Pang ◽  
Zhijian Zhang ◽  
Yufeng Wang ◽  
Shengfen Wang ◽  
Yuanyuan Song ◽  
...  
Keyword(s):  
Risk Factors ◽  
Mycobacterium Tuberculosis ◽  
Population Structure ◽  
Drug Susceptibility ◽  
Beijing Genotype ◽  
Content Type ◽  
The Past ◽  
Resistant Tuberculosis ◽  
Prevalence Trends ◽  
Significant Difference

ABSTRACT We investigated the prevalence, trends, and risk factors for pyrazinamide (PZA) and moxifloxacin (MOX) resistance among tuberculosis (TB) cases in China and also analyzed the population structure of Mycobacterium tuberculosis strains. All the M. tuberculosis strains enrolled in this study were collected from the national TB prevalence surveys. Each strain was genotyped by analyzing the regions of RD105 and IS6110 in the NTF region. The Bactec MGIT 960 system was used to detect the drug susceptibility of M. tuberculosis isolates to PZA and MOX. Based on the genotyping results, 241 (66.4%) strains were classified as Beijing genotype in 2000, which was significantly lower than in 2010 (76.2%, P < 0.01). The proportion of the modern Beijing genotype increased significantly from 49.6% in 2000 to 68.1% in 2010 (P < 0.01), while no significant difference was observed in the rate of ancient Beijing genotype between 2000 and 2010 (P = 0.676). In addition, we found that the proportion of PZA resistance in 2010 (15.0%) was significantly higher than that in 2000 (9.6%, P = 0.04). For MOX, there were more MOX-resistant isolates detected in 2010 (7.7%) than in 2000 (3.0%). In conclusion, our data demonstrate that the Beijing genotype was the predominant M. tuberculosis lineage during the past decade. The proportion of Beijing genotype isolates significantly increased from 2000 to 2010, largely due to an increase in the modern Beijing sublineage. In addition, resistance to PZA and MOX increased significantly in China between 2000 and 2010.

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 Disparities in Capreomycin Resistance Levels Associated with therrsA1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

2014 ◽  
Vol 59 (1) ◽  
pp. 444-449 ◽  
Author(s):  
Analise Z. Reeves ◽  
Patricia J. Campbell ◽  
Melisa J. Willby ◽  
James E. Posey
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Strong Predictor ◽  
Critical Concentration ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Second Line ◽  
Content Type

ABSTRACTAs the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. TherrsA1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between therrsA1401G mutation and CAP resistance, with up to 40% ofrrsA1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring therrsA1401G mutation and found that the CAP MICs ranged from 8 μg/ml to 40 μg/ml. These results were drastically different from engineered A1401G mutants generated in isogenicMycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 μg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 μg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

scholarly journals A Diagnostic Algorithm To Investigate Pyrazinamide and Ethambutol Resistance in Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Low-Incidence Setting

2018 ◽  
Vol 63 (2) ◽  
pp. e01798-18 ◽  
Author(s):  
Söenke Andres ◽  
Matthias I. Gröschel ◽  
Doris Hillemann ◽  
Matthias Merker ◽  
Stefan Niemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sanger Sequencing ◽  
Rational Design ◽  
Clinical Decision Making ◽  
Susceptibility Testing ◽  
Diagnostic Algorithm ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Content Type ◽  
Phenotypic Resistance

ABSTRACT Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol.

scholarly journals Sensititre MycoTB Plate Compared to Bactec MGIT 960 for First- and Second-Line Antituberculosis Drug Susceptibility Testing in Tanzania: a Call To Operationalize MICs

2015 ◽  
Vol 59 (11) ◽  
pp. 7104-7108 ◽  
Author(s):  
Scott K. Heysell ◽  
Suporn Pholwat ◽  
Stellah G. Mpagama ◽  
Saumu J. Pazia ◽  
Happy Kumburu ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Acquired Resistance ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Second Line ◽  
Drug Resistant Tuberculosis ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Extensively Drug Resistant

ABSTRACTMIC testing forMycobacterium tuberculosisis now commercially available. Drug susceptibility testing by the MycoTB MIC plate has not been directly compared to that by the Bactec MGIT 960. We describe a case of extensively drug-resistant tuberculosis (XDR-TB) in Tanzania where initial MIC testing may have prevented acquired resistance. From testing on archived isolates, the accuracy with the MycoTB plate was >90% for important first- and second-line drugs compared to that with the MGIT 960, and clinically useful quantitative interpretation was also provided.

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 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 Impact of gyrB and eis Mutations in Improving Detection of Second-Line-Drug Resistance among Mycobacterium tuberculosis Isolates from Georgia

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
N. Bablishvili ◽  
N. Tukvadze ◽  
E. Shashkina ◽  
B. Mathema ◽  
N. R. Gandhi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
First Generation ◽  
High Specificity ◽  
Kanamycin Resistance ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Resistant Tuberculosis ◽  
Country Of Georgia ◽  
Low Sensitivity ◽  
Resistance Detection

ABSTRACT The country of Georgia has a high burden of multi- and extensively drug-resistant tuberculosis (XDR-TB). To evaluate whether mutations in gyrB and eis genes increased the sensitivity of detection of phenotypic resistance to ofloxacin and kanamycin or capreomycin compared to use of the first-generation MTBDRsl assay alone, which tests for mutations in gyrA and rrs genes, a retrospective study of stored Mycobacterium tuberculosis isolates was performed. All isolates underwent DNA sequencing of resistance-determining regions. Among 112 M. tuberculosis isolates with DNA extraction data, targeted sequencing was successfully performed for each gene as follows: for gyrA, 98% sensitivity; for gyrB, 96%; for rrs, 93%; for the eis gene and its promoter, 93%. The specificity and hence the positive predictive value of gyrA and gyrB mutations for detecting ofloxacin resistance were 100%. The addition of gyrB mutations increased the sensitivity of phenotypic ofloxacin resistance detection by 13% (75% to 88%). All rrs resistance-conferring mutations were A1401G, and this mutation had low sensitivity (40% and 18%) and high specificity (95% and 100%) in predicting phenotypic capreomycin and kanamycin resistance, respectively. The eis C-14T mutation increased the sensitivity of phenotypic kanamycin resistance detection by 9% (18% to 27%) and was found solely in kanamycin phenotypic resistance isolates. Our data showed that the inclusion of eis C-14T and gyrB mutations in addition to rrs and gyrA mutations improves the sensitivity of detection of phenotypic ofloxacin and kanamycin resistance, respectively.

scholarly journals Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Bin Liang ◽  
Yaoju Tan ◽  
Zi Li ◽  
Xueshan Tian ◽  
Chen Du ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Melting Curve ◽  
Digital Pcr ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Melting Curve Analysis ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Highly Sensitive Detection

ABSTRACT Detection of heteroresistance of Mycobacterium tuberculosis remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less-abundant mutants through selective clamping of the wild type in mixed populations. The singleplex DeepMelt assay detected 0.01% katG S315T in 105 M. tuberculosis genomes/μl. The multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 104 M. tuberculosis genomes/μl. The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 precharacterized clinical isolates. Using the 1% proportion method as the gold standard, the sensitivity was found to be increased from 93.6% (176/188, 95% confidence interval [CI] = 89.2 to 96.3%) to 95.7% (180/188, 95% CI = 91.8 to 97.8%) compared to the MeltPro TB/INH assay. Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3% (20/24, 95% CI = 64.2 to 93.3%) to 91.7% (22/24, 95% CI = 74.2 to 97.7%). In both cases, the specificity remained nearly unchanged. All heteroresistant samples newly identified by the DeepMelt TB/INH assay were confirmed by DNA sequencing and even partially by digital PCR. The DeepMelt assay may fill the gap between current genotypic and phenotypic drug susceptibility testing for detecting drug-resistant tuberculosis patients.

scholarly journals Resazurin Microtiter Assay Plate Testing of Mycobacterium tuberculosis Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method

2003 ◽  
Vol 47 (11) ◽  
pp. 3616-3619 ◽  
Author(s):  
Anandi Martin ◽  
Mirtha Camacho ◽  
Françoise Portaels ◽  
Juan Carlos Palomino
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Second Line ◽  
Aminosalicylic Acid ◽  
Resistant Tuberculosis ◽  
Proportion Method ◽  
Susceptibility Tests ◽  
Visual Reading ◽  
Microtiter Assay

ABSTRACT The emergence of multidrug-resistant tuberculosis calls for new, rapid drug susceptibility tests. We have tested 150 Mycobacterium tuberculosis isolates against the second-line drugs ethionamide, kanamycin, capreomycin, ofloxacin, and para-aminosalicylic acid by the colorimetric resazurin microtiter assay and the proportion method. By visual reading, MICs were obtained after 8 days. A very good correlation between results by the colorimetric resazurin microtiter assay and the proportion method was obtained. The colorimetric resazurin microtiter assay is inexpensive, rapid, and simple to perform, and implementation of the assay is feasible for low-resource countries.

Sign in / Sign up

Export Citation Format

Share Document