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      Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use

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          Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

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          Author and article information

          Role: Editor
          J Clin Microbiol
          J. Clin. Microbiol
          Journal of Clinical Microbiology
          American Society for Microbiology (1752 N St., N.W., Washington, DC )
          2 November 2016
          28 December 2016
          January 2017
          : 55
          : 1
          : 183-198
          [a ]Department of Medicine, New Jersey Medical School, Newark, New Jersey, USA
          [b ]Cepheid Inc., Sunnyvale, California, USA
          [c ]Cepheid Inc., Bothell, Washington, USA
          [d ]Henan Provincial Chest Hospital, Zhengzhou, Henan Province, China
          [e ]Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of Medical Microbiology, School of Basic Medical Sciences, Fudan University, Shanghai, China
          [f ]Sino-US International Research Center of Tuberculosis, Zhengzhou, China
          [g ]Henan Public Health Clinical Center, Zhengzhou, China
          [h ]Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA
          [i ]Institute of Infectious Disease and Molecular Medicine, Department of Pathology, University of Cape Town, Cape Town, South Africa
          [j ]Department of Microbiology, International Tuberculosis Research Center, Changwon, Gyeongsnag, Republic of Korea
          Carter BloodCare and Baylor University Medical Center
          Author notes
          Address correspondence to Soumitesh Chakravorty, chakraso@ 123456njms.rutgers.edu .

          Citation Chakravorty S, Roh SS, Glass J, Smith LE, Simmons AM, Lund K, Lokhov S, Liu X, Xu P, Zhang G, Via LE, Shen Q, Ruan X, Yuan X, Zhu HZ, Viazovkina E, Shenai S, Rowneki M, Lee JS, Barry CE, III, Gao Q, Persing D, Kwiatkawoski R, Jones M, Gall A, Alland D. 2017. Detection of isoniazid-, fluoroquinolone-, amikacin-, and kanamycin-resistant tuberculosis in an automated, multiplexed 10-color assay suitable for point-of-care use. J Clin Microbiol 55:183–198. https://doi.org/10.1128/JCM.01771-16.

          PMC5228229 PMC5228229 5228229 01771-16
          Copyright © 2016 American Society for Microbiology.
          Page count
          supplementary-material: 1, Figures: 6, Tables: 2, Equations: 0, References: 57, Pages: 16, Words: 10082
          Mycobacteriology and Aerobic Actinomycetes
          Custom metadata
          January 2017


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