scholarly journals GenoType NTM-DR Performance Evaluation for Identification of Mycobacterium avium Complex and Mycobacterium abscessus and Determination of Clarithromycin and Amikacin Resistance

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Hee Jae Huh ◽  
Su-Young Kim ◽  
Hyang Jin Shim ◽  
Dae Hun Kim ◽  
In Young Yoo ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Drug Susceptibility ◽  
Rapid Identification ◽  
Drug Susceptibility Testing ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Resistance Mutations ◽  
Content Type ◽  
Clarithromycin Resistance ◽  
Reference Strains

ABSTRACT We evaluated the GenoType NTM-DR (NTM-DR) line probe assay for identifying Mycobacterium avium complex (MAC) species and Mycobacterium abscessus subspecies and for determining clarithromycin and amikacin resistance. Thirty-eight reference strains and 145 clinical isolates (58 MAC and 87 M. abscessus isolates), including 54 clarithromycin- and/or amikacin-resistant strains, were involved. The performance of the NTM-DR assay in rapid identification was evaluated by comparison with results of multigene sequence-based typing, whereas performance in rapid detection of clarithromycin and amikacin resistance was evaluated by comparison with sequencing of the erm(41), rrl, and rrs genes and drug susceptibility testing (DST). The accuracies of MAC and M. abscessus (sub)species identification were 92.1% (35/38) and 100% (145/145) for the 38 reference strains and 145 clinical isolates, respectively. Three MAC strains other than M. intracellulare were found to cross-react with the M. intracellulare probe in the assay. Regarding clarithromycin resistance, NTM-DR detected rrl mutations in 52 isolates and yielded 99.3% (144/145) and 98.6% (143/145) concordant results with sequencing and DST, respectively. NTM-DR sensitivity and specificity in the detection of clarithromycin resistance were 96.3% (52/54) and 100% (91/91), respectively. The NTM-DR yielded accurate erm(41) genotype results for all 87 M. abscessus isolates. Regarding amikacin resistance, NTM-DR detected rrs mutations in five isolates and yielded 99.3% (144/145) and 97.9% (142/145) concordant results with sequencing and DST, respectively. Our results indicate that the NTM-DR assay is a straightforward and accurate approach for discriminating MAC and M. abscessus (sub)species and for detecting clarithromycin and amikacin resistance mutations and that it is a useful tool in the clinical setting.

scholarly journals Molecular Drug Susceptibility Testing and Genotyping of Mycobacterium leprae Strains from South America

2011 ◽  
Vol 55 (6) ◽  
pp. 2971-2973 ◽  
Author(s):  
Pushpendra Singh ◽  
Philippe Busso ◽  
Alberto Paniz-Mondolfi ◽  
Nacarid Aranzazu ◽  
Marc Monot ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Mutation ◽  
Mycobacterium Leprae ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Polymorphism Analysis ◽  
South American ◽  
Resistance Mutations ◽  
Single Nucleotide ◽  
Content Type

ABSTRACTPossible drug resistance inMycobacterium lepraestrains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

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 The Contribution of Efflux Pumps in Mycobacterium abscessus Complex Resistance to Clarithromycin

Antibiotics ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 153 ◽  
Author(s):  
Júlia S. Vianna ◽  
Diana Machado ◽  
Ivy B. Ramis ◽  
Fábia P. Silva ◽  
Dienefer V. Bierhals ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Mycobacterium Abscessus ◽  
New Drugs ◽  
Clarithromycin Resistance ◽  
Mycobacterium Abscessus Complex

The basis of drug resistance in Mycobacterium abscessus is still poorly understood. Nevertheless, as seen in other microorganisms, the efflux of antimicrobials may also play a role in M. abscessus drug resistance. Here, we investigated the role of efflux pumps in clarithromycin resistance using nine clinical isolates of M. abscessus complex belonging to the T28 erm(41) sequevar responsible for the inducible resistance to clarithromycin. The strains were characterized by drug susceptibility testing in the presence/absence of the efflux inhibitor verapamil and by genetic analysis of drug-resistance-associated genes. Efflux activity was quantified by real-time fluorometry. Efflux pump gene expression was studied by RT-qPCR upon exposure to clarithromycin. Verapamil increased the susceptibility to clarithromycin from 4- to ≥64-fold. The efflux pump genes MAB_3142 and MAB_1409 were found consistently overexpressed. The results obtained demonstrate that the T28 erm(41) polymorphism is not the sole cause of the inducible clarithromycin resistance in M. abscessus subsp. abscessus or bolletii with efflux activity providing a strong contribution to clarithromycin resistance. These data highlight the need for further studies on M. abscessus efflux response to antimicrobial stress in order to implement more effective therapeutic regimens and guidance in the development of new drugs against these bacteria.

scholarly journals Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

2011 ◽  
Vol 56 (3) ◽  
pp. 1253-1257 ◽  
Author(s):  
J. Werngren ◽  
E. Sturegård ◽  
P. Juréen ◽  
K. Ängeby ◽  
S. Hoffner ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Wild Type ◽  
First Line ◽  
Content Type ◽  
Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

scholarly journals Laboratory Evaluation of a Lateral-Flow Cell for Molecular Detection of First-Line and Second-Line Antituberculosis Drug Resistance

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Donald G. Catanzaro ◽  
Rebecca E. Colman ◽  
Yvonne Linger ◽  
Sophia B. Georghiou ◽  
Alexander V. Kukhtin ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Drug Susceptibility ◽  
Flow Cell ◽  
Drug Susceptibility Testing ◽  
Lateral Flow ◽  
Laboratory Evaluation ◽  
Second Line ◽  
Resistance Mutations ◽  
Content Type ◽  
Resistance Patterns

ABSTRACT Despite the WHO’s call for universal drug susceptibility testing for all patients being evaluated for tuberculosis (TB), a lack of rapid diagnostic tests which can fully describe TB resistance patterns is a major challenge in ensuring that all persons diagnosed with drug-resistant TB are started on an appropriate treatment regime. We evaluated the accuracy of the Akonni Biosystems XDR-TB TruArray and lateral-flow cell (XDR-LFC), a novel multiplex assay to simultaneously detect mutations across seven genes that confer resistance to both first- and second-line anti-TB drugs. The XDR-LFC includes 271 discrete three-dimensional gel elements with target-specific probes for identifying mutations in katG, inhA promoter, and ahpC promoter (isoniazid), rpoB (rifampin), gyrA (fluoroquinolones), rrs and eis promoter (kanamycin), and rrs (capreomycin and amikacin). We evaluated XDR-LFC performance with 87 phenotypically and genotypically characterized clinical Mycobacterium tuberculosis isolates. The overall assay levels of accuracy for mutation detection in specific genes were 98.6% for eis promoter and 100.0% for the genes katG, inhA promoter, ahpC promoter, rpoB, gyrA, and rrs. The sensitivity and specificity against phenotypic reference were 100% and 100% for isoniazid, 98.4% and 50% for rifampin (specificity increased to 100% once the strains with documented low-level resistance mutations in rpoB were excluded), 96.2% and 100% for fluoroquinolones, 92.6% and 100% for kanamycin, 93.9% and 97.4% for capreomycin, and 80% and 100% for amikacin. The XDR-LFC solution appears to be a promising new tool for accurate detection of resistance to both first- and second-line anti-TB drugs.

scholarly journals In Vitro Activity of Bedaquiline and Delamanid against Nontuberculous Mycobacteria, Including Macrolide-Resistant Clinical Isolates

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Dae Hun Kim ◽  
Byung Woo Jhun ◽  
Seong Mi Moon ◽  
Su-Young Kim ◽  
Kyeongman Jeon ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Nontuberculous Mycobacteria ◽  
Mycobacterium Abscessus ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
In Vitro Activity ◽  
Mycobacterium Kansasii ◽  
Antimicrobial Drugs ◽  
Content Type

ABSTRACT We evaluated the in vitro activities of the antimicrobial drugs bedaquiline and delamanid against the major pathogenic nontuberculous mycobacteria (NTM). Delamanid showed high MIC values for all NTM except Mycobacterium kansasii. However, bedaquiline showed low MIC values for the major pathogenic NTM, including Mycobacterium avium complex, Mycobacterium abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. kansasii. Bedaquiline also had low MIC values with macrolide-resistant NTM strains and warrants further investigation as a potential antibiotic for NTM treatment.

scholarly journals Mutations ingyrAandgyrBin Moxifloxacin-ResistantMycobacterium aviumComplex andMycobacterium abscessusComplex Clinical Isolates

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Su-Young Kim ◽  
Byung Woo Jhun ◽  
Seong Mi Moon ◽  
Sun Hye Shin ◽  
Kyeongman Jeon ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Content Type

ABSTRACTData on the frequency ofgyrAandgyrBmutations in fluoroquinolone-resistant isolates of theMycobacterium aviumcomplex (MAC) and theMycobacterium abscessuscomplex (MABC) are limited. In our analysis, we did not find any resistance-associated mutations ingyrAorgyrBin 105 MAC or MABC clinical isolates, including 72 moxifloxacin-resistant isolates. Our findings suggest that mechanisms other thangyrAandgyrBmutations contribute to moxifloxacin resistance in these organisms.

scholarly journals Evaluation of the MGIT 960/EpiCenter TB eXiST system for drug susceptibility testing for Mycobacterium abscessus group

2020 ◽  
Author(s):  
Robert D Arbeit ◽  
Natalia de CARVALHO ◽  
Sylvia LEÃO ◽  
Erica CHIMARA
Keyword(s):  
Internal Control ◽  
Reference Strain ◽  
Impact Resistance ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Reference Laboratory ◽  
Inducible Resistance ◽  
Technical Factors

Abstract BackgroundDrug susceptibility test (DST) of the Mycobacterium abscessus group (MAG) and other rapidly growing nontuberculous mycobacteria by conventional microplate techniques is complicated due to inducible resistance to clarithromycin and other technical factors. This study evaluated the application of the BACTEC MGIT 960/Epicenter TB eXiST for DST of MAG clinical isolates. MethodsM. abscessus ATCC19977 was used as the reference strain for the standardizing the DST by MGIT 960 and as the internal control for testing of 31 clinical isolates tests submitted to a reference laboratory for DST and confirmed as MAG. Clarithromycin genotyping was performed for the loci in the rrl and erm (41) genes known to impact resistance phenotype. ResultsThe 31 MAG isolates included 14 M. abscessus , 8 M. massiliense , and 9 M. bolletii . Using conventional microplate technique according to CLSI guideline, the isolates had a high percentage of resistance for cefoxitin (93.5%) and imipenem (100%), and sensitivity for amikacin (96.7%). Comparing microplate and MGIT 960 results across those 93 pairs of results (31 isolates x 3 antibiotics), 73 (80.6%) were concordant and the remaining 18 (19.4%) represented minor errors; there were no major or very major errors. Concordance was 100% for amikacin, 84% for imipenem and 58% for cefoxitin. Clarithromycin DST by microplate identified 14 isolates as susceptible (all susceptible by MGIT 960), 3 isolates as resistant after 3 days incubation, and 14 isolates demonstrating inducible resistance from Day 5 through 14. Among the latter isolates, MGIT 960 reported 8 as resistant and 6 as intermediate, without modifications to the protocol developed. For all isolates, the observed clarithromycin susceptibility phenotypes were consistent with the genotypes. ConclusionThe present study is the first description of a DST protocol for MAG isolates using the MGIT 960/Epicenter TB eXiST system. The protocol developed provided highly reliable results based on direct comparison with the conventional microplate method, including, without further modification, detection of isolates with inducible-resistance to clarithromycin. Laboratories using MGIT 960 for DST of other mycobacteria may find benefit to incorporating MAG into their routine.

scholarly journals Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Suporn Pholwat ◽  
Jie Liu ◽  
Suzanne Stroup ◽  
Jean Gratz ◽  
Sayera Banu ◽  
...  
Keyword(s):  
High Resolution ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Resistance Mutations ◽  
High Resolution Melt ◽  
Content Type ◽  
High Resolution Melt Analysis ◽  
Taqman Array

ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.

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