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Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing

2015; Public Library of Science; Volume: 10; Issue: 5 Linguagem: Inglês

10.1371/journal.pone.0126626

1932-6203

Rebecca E. Colman, James M. Schupp, Nathan Hicks, David Smith, Jordan L. Buchhagen, Faramarz Valafar, Valeriu Crudu, Elena Romancenco, Ecaterina Noroc, Lynn Jackson, Donald G. Catanzaro, Timothy C. Rodwell, Antonino Catanzaro, Paul Keim, David M. Engelthaler,

Infectious Diseases and Tuberculosis

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.