Development and in-use evaluation of a novel Luminex MicroPlex microsphere-based (TRIOL) assay for simultaneous identification of Mycobacterium tuberculosis and detection of first-line and second-line anti-tuberculous drug resistance in China

2016 ◽  
Vol 70 (4) ◽  
pp. 342-349 ◽  
Author(s):  
Feifei Yin ◽  
Jasper Fuk-Woo Chan ◽  
Qixuan Zhu ◽  
Ruijia Fu ◽  
Jonathan Hon-Kwan Chen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Open Architecture ◽  
Rifampicin Resistance ◽  
Lower Sensitivity ◽  
Second Line ◽  
First Line ◽  
Microbial Identification ◽  
Multiple Drugs ◽  
Resistance Detection

AimsRapid and accurate diagnostic assays with simultaneous microbial identification and drug resistance detection are essential for optimising treatment and control of tuberculosis.MethodsWe developed a novel multiplex (TRIOL, Tuberculosis-Rifampicin-Isoniazid-Ofloxacin-Luminex) assay using the Luminex xMAP system that simultaneously identifies Mycobacterium tuberculosis and detects resistance to first-line and second-line anti-tuberculous drugs, and compared its performance with that by PCR sequencing, using phenotypic drug susceptibility testing as the gold standard.ResultsIdentification of M. tuberculosis by the TRIOL assay was highly sensitive (100%) and specific (100%). The overall drug-specific specificities were excellent (100%). The overall sensitivity of the TRIOL assay was lower than that of the PCR-sequencing assays (72.4% vs 82.8%) because of a lower sensitivity of detecting rifampicin resistance (71.4% vs 92.9%). The sensitivity of detecting isoniazid and ofloxacin resistance was as good as the PCR-sequencing assays. Importantly, the TRIOL assay did not miss any mutations that were included in the assay. All of the resistant isolates that were missed had uncommon mutations or unknown resistance mechanisms that were not included in the assay.ConclusionsThe TRIOL assay has higher throughput, lower cost and is less labour intensive than the PCR-sequencing assays. The TRIOL assay is advantageous in having the capability to detect resistance to multiple drugs and an open-architecture system that allows addition of more specific primers to detect uncommon mutations. Inclusion of additional primers for the identification of non-tuberculous mycobacteria, spoligotyping and improvement of rifampicin resistance detection would enhance the use of the TRIOL assay in future clinical and epidemiological studies.

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 Increasing prevalence of resistance to second-line drugs among multidrug-resistant Mycobacterium tuberculosis isolates in Kuwait

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Noura M. Al-Mutairi ◽  
Suhail Ahmad ◽  
Eiman Mokaddas
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Mutation Detection ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Genetic Mutations ◽  
Second Line ◽  
Injectable Drugs ◽  
Mdr Tb ◽  
Phenotypic Susceptibility

AbstractMolecular methods detect genetic mutations associated with drug resistance. This study detected resistance-conferring mutations in gyrA/gyrB for fluoroquinolones and rrs/eis genes for second-line injectable drugs (SLIDs) among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Kuwait. Fifty pansusceptible M. tuberculosis and 102 MDR-TB strains were tested. Phenotypic susceptibility testing was performed by MGIT 960 system using SIRE drug kit. GenoType MTBDRsl version 1 (gMTBDRslv1) and GenoType MTBDRsl version 2 (gMTBDRslv2) tests were used for mutation detection. Results were validated by PCR-sequencing of respective genes. Fingerprinting was performed by spoligotyping. No mutations were detected in pansusceptible isolates. gMTBDRslv1 detected gyrA mutations in 12 and rrs mutations in 8 MDR-TB isolates. gMTBDRsl2 additionally detected gyrB mutations in 2 and eis mutation in 1 isolate. Mutations in both gyrA/gyrB and rrs/eis were not detected. gMTBDRslv1 also detected ethambutol resistance-conferring embB mutations in 59 isolates. Although XDR-TB was not detected, frequency of resistance-conferring mutations for fluoroquinolones or SLIDs was significantly higher among isolates collected during 2013–2019 versus 2006–2012. Application of both tests is warranted for proper management of MDR-TB patients in Kuwait as gMTBDRslv2 detected resistance to fluoroquinolones and/or SLIDs in 3 additional isolates while gMTBDRslv1 additionally detected resistance to ethambutol in 58% of MDR-TB isolates.

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 Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

2014 ◽  
Vol 59 (1) ◽  
pp. 414-420 ◽  
Author(s):  
Kanchan Ajbani ◽  
Shou-Yean Grace Lin ◽  
Camilla Rodrigues ◽  
Duylinh Nguyen ◽  
Francine Arroyo ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
The Philippines ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Second Line ◽  
Additional Advantage ◽  
Content Type ◽  
Extensively Drug Resistant

ABSTRACTReliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance inMycobacterium tuberculosisisolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identifyM. tuberculosis(via the IS6110marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets includedkatG, theinhApromoter and theahpC-oxyRintergenic region for isoniazid (INH) resistance; therpoBcore region for rifampin (RIF) resistance;gyrAfor fluoroquinolone (FQ) resistance; andrrsfor amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

scholarly journals Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e43240 ◽  
Author(s):  
Indra Bergval ◽  
Sarah Sengstake ◽  
Nadia Brankova ◽  
Viktoria Levterova ◽  
Edgar Abadía ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Species Identification ◽  
Resistance Detection

scholarly journals Clinical evaluation of the Xpert MTB/XDR assay for rapid detection of isoniazid, fluoroquinolone, ethionamide and second-line drug resistance: A cross-sectional multicentre diagnostic accuracy study

2021 ◽  
Author(s):  
Adam Penn-Nicholson ◽  
Sophia B Georghiou ◽  
Nelly Ciobanu ◽  
Mubin Kazi ◽  
Manpreet Bhalla ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Diagnostic Accuracy ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Foreign Affairs ◽  
Second Line ◽  
Reference Standard ◽  
Composite Reference Standard ◽  
Resistance Detection

Background The WHO End TB Strategy requires universal drug susceptibility testing and treatment of all people with tuberculosis. However, available second-line diagnostic tools are cumbersome and require sophisticated laboratory infrastructure, and ultimately less than half of those with drug-resistant tuberculosis receive appropriate treatment. Xpert MTB/XDR was developed to help overcome these limitations. Methods We assessed the diagnostic accuracy of sputum-based Xpert MTB/XDR for isoniazid, fluoroquinolone, ethionamide and second-line injectable resistance detection in adults with an Xpert MTB/RIF or Ultra Mycobacterium tuberculosis-positive result against a composite reference standard of phenotypic drug-susceptibility testing and whole genome sequencing (NCT03728725). Participants with pulmonary tuberculosis symptoms and ≥1 risk factor for drug resistance were consecutively enrolled between four clinical sites in India, Moldova and South Africa. Findings Between 31 July 2019 and 21 March 2020, we enrolled 710 patients, of which 611 (86.1%) had results from index and composite reference standard tests and were included in analysis. The sensitivity of Xpert MTB/XDR was 94% for isoniazid, 95% for fluoroquinolones, 54% for ethionamide, 73% for amikacin, 86% for kanamycin, and 61% for capreomycin resistance detection. Specificity was 98-100% for all drugs. Performance was equivalent to line-probe assays. The non-determinate rate of Xpert MTB/XDR was 2.96%. Interpretation This first prospective, multicentre clinical study of the Xpert MTB/XDR assay demonstrated high diagnostic test accuracy, meeting target product profile criteria for a next-generation drug susceptibility test. Funding German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.

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