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      Correlation between the BACTEC MGIT 960 culture system with Genotype MTBDR plus and TB-SPRINT in multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

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          Abstract

          BACKGROUND

          The accurate detection of multidrug-resistant tuberculosis (MDR-TB) is critical for the application of appropriate patient treatment and prevention of transmission of drug-resistant Mycobacterium tuberculosis isolates. The goal of this study was to evaluate the correlation between phenotypic and molecular techniques for drug-resistant tuberculosis diagnostics. Molecular techniques used were the line probe assay genotype MTBDR plus and the recently described tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT) bead-based assay. Conventional drug susceptibility testing (DST) was done on a BACTEC TM MGIT 960 TB.

          METHOD

          We studied 80 M. tuberculosis complex (MTC) clinical isolates from Minas Gerais state, of which conventional DST had classified 60 isolates as MDR and 20 as drug susceptible.

          FINDINGS

          Among the 60 MDR-TB isolates with MGIT as a reference, sensitivity, specificity, accuracy, and kappa for rifampicin (RIF) resistance using TB-SPRINT and MTBDR plus, were 96.7% versus 93.3%, 100.0% versus 100.0%, 97.5% versus 95.0% and 0.94 versus 0.88, respectively. Similarly, the sensitivity, specificity, accuracy, and kappa for isoniazid (INH) resistance were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively. Finally, the sensitivity, specificity, accuracy, and kappa for MDR-TB were 85.0% and 83.3%, 100.0% and 100.0%, 88.8% and 87.5% and 0.74 and 0.71 for both tests, respectively.

          MAIN CONCLUSIONS

          Both methods exhibited a good correlation with the conventional DST. We suggest estimating the cost-effectiveness of MTBDR plus and TB-SPRINT in Brazil.

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          Most cited references30

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          Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis.

          The 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), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex 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% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. 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 the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.
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            Mechanisms of drug resistance in Mycobacterium tuberculosis.

            The increasing emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) in the era of human immunodeficiency virus (HIV) infection presents a major threat to effective control of TB. Drug resistance in Mycobacterium tuberculosis arises from spontaneous chromosomal mutations at low frequency. Clinical drug-resistant TB largely occurs as a result of man-made selection during disease treatment of these genetic alterations through erratic drug supply, suboptimal physician prescription and poor patient adherence. Molecular mechanisms of drug resistance have been elucidated for the major first- and second-line drugs rifampicin, isoniazid, pyrazinamide, ethambutol, the aminoglycosides and the fluoroquinolones. The relationship between drug resistance in M. tuberculosis strains and their virulence/transmissibility needs to be further investigated. Understanding the mechanisms of drug resistance in M. tuberculosis would enable the development of rapid molecular diagnostic tools and furnish possible insights into new drug development for the treatment of TB.
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              Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

              Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.
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                Author and article information

                Journal
                Mem Inst Oswaldo Cruz
                Mem. Inst. Oswaldo Cruz
                mioc
                Memórias do Instituto Oswaldo Cruz
                Instituto Oswaldo Cruz, Ministério da Saúde
                0074-0276
                1678-8060
                November 2017
                November 2017
                : 112
                : 11
                : 769-774
                Affiliations
                [1 ]Universidade Federal de Minas Gerais, Faculdade de Medicina, Departamento de Clínica Médica, Programa de Pós-Graduação em Infectologia e Medicina Tropical, Belo Horizonte, MG, Brasil
                [2 ]Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular Aplicada a Micobactéria, Rio de Janeiro, RJ, Brasil
                [3 ]Universidade Federal de Minas Gerais, Faculdade de Farmácia, Departamento de Farmácia Social, Laboratório de Biologia Molecular e Saúde Pública, Belo Horizonte, MG, Brasil
                [4 ]Fundação Ezequiel Dias, Belo Horizonte, MG, Brasil
                [5 ]Centre Muraz, Bobo-Dioulasso, Burkina Faso
                [6 ]Institut for Integrative Cell Biology, UMR9198 CEA-CNRS-UPSaclay, Orsay, France
                [7 ]University Paris-Sud, Beamedex SAS, Orsay, France
                Author notes
                [+ ] Corresponding author: silvanaspindola@ 123456gmail.com

                AUTHORS’ CONTRIBUTION

                NGTD, WSC, CS and SSM - Conceived and designed the experiments; NGTD, INA, LJA and ADG - performed the experiments; NGTD, HMG, INA, ADG, MKG, GR, CS and SSM - analysed the data; NGTD, PNS, HMG, GR, CS and SSM - wrote the paper.

                Article
                0074-02760170062
                10.1590/0074-02760170062
                5661900
                29091137
                2f8fee1f-2363-4550-956c-9fcbfffd5fcc

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 10 February 2017
                : 13 June 2017
                Page count
                Figures: 0, Tables: 2, Equations: 0, References: 28, Pages: 6
                Categories
                Article

                tuberculosis,multidrug-resistant,genotype,molecular diagnosis

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