The incidence of extensively drug resistant (XDR) Mycobacterium tuberculosis (TB) is increasing at an alarming rate. Early and rapid detection of XDR TB could be crucial to disease management by alerting physicians to use salvage therapy and to institute special infection control procedures. This proposal seeks to leverage several successful programs in molecular diagnostics, sample processing, TB genetics and TB clinical trials at The New Jersey Medical School-UMDNJ, the NIH, and the biotechnology company Cepheid and the International Tuberculosis Research Center/National Masan Tuberculosis Hospital (NMTH) in Masan, Korea. Our collaborative goal is to transform detection of XDR TB from a months-long task into a 90- minute assay that can be performed directly on clinical sputum samples. We will develop a highly multiplexed XDR assay that uses a novel type of "sloppy molecular beacon" to perform multiplex melting temperature analysis and mutation detection. Reasons for discordances between conventional drug susceptibility testing and molecular assays will be investigated and the results used to make further assay improvements. An optimized assay incorporating all important XDR mutations will be tested, reduced to a format compatible with a simple sample-processing cartridge and then piloted on clinical samples in Masan.
The specific aims of this proposal are to: 1) Develop a multiplexed molecular beacon-PCR assay to rapidly identify the common causes of TB resistance to FQs and injectable antibiotics. 2) Test the new assay on a library of TB samples, identify the causes of discordant results, and improve the assay coverage using same color multiplexing. 3) Reformat the XDR assay so that it can be performed directly on clinical sputum samples and detect XDR within two hours. Our overall goal is to transform XDR detection and management.
This project will also lead to a very rapid test to detect highly drug resistant tuberculosis so that it can be treated more rapidly and so that patients with this disease can be quickly isolated before they infect others.
|Lingaraju, Subramanya; Rigouts, Leen; Gupta, Aditi et al. (2016) Geographic Differences in the Contribution of ubiA Mutations to High-Level Ethambutol Resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 60:4101-5|
|Roh, Sandy S; Smith, Laura E; Lee, Jong Seok et al. (2015) Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance. PLoS One 10:e0126257|
|Chakravorty, Soumitesh; Lee, Jong Seok; Cho, Eun Jin et al. (2015) Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testin J Clin Microbiol 53:43-51|
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|Wilson, Regina; Kumar, Pradeep; Parashar, Vijay et al. (2013) Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat Chem Biol 9:499-506|
|Banada, Padmapriya P; Koshy, Ranie; Alland, David (2013) Detection of Mycobacterium tuberculosis in blood by use of the Xpert MTB/RIF assay. J Clin Microbiol 51:2317-22|
|Chakravorty, Soumitesh; Kothari, Harsheel; Aladegbami, Bola et al. (2012) Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding. J Clin Microbiol 50:2194-202|
|Blakemore, Robert; Nabeta, Pamela; Davidow, Amy L et al. (2011) A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay. Am J Respir Crit Care Med 184:1076-84|
|Chakravorty, Soumitesh; Aladegbami, Bola; Thoms, Kimberley et al. (2011) Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay. J Clin Microbiol 49:932-40|
|Safi, Hassan; Fleischmann, Robert D; Peterson, Scott N et al. (2010) Allelic exchange and mutant selection demonstrate that common clinical embCAB gene mutations only modestly increase resistance to ethambutol in Mycobacterium tuberculosis. Antimicrob Agents Chemother 54:103-8|
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