title: Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.
abstract: 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.
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Colman
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
authors.forename: James M
authors.initials: JM
authors.lastname: Schupp
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Hicks
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
authors.forename: David E
authors.initials: DE
authors.lastname: Smith
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Buchhagen
authors.affiliation: San Diego State University, San Diego, CA, United States of America.
authors.forename: Faramarz
authors.initials: F
authors.lastname: Valafar
authors.affiliation: Phthisiopneumology Institute (PPI), Chisinau, Republic of Moldova.
authors.forename: Valeriu
authors.initials: V
authors.lastname: Crudu
authors.affiliation: University of California San Diego, San Diego, CA, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Romancenco
authors.affiliation: Phthisiopneumology Institute (PPI), Chisinau, Republic of Moldova.
authors.forename: Ecaterina
authors.initials: E
authors.lastname: Noroc
authors.affiliation: University of California San Diego, San Diego, CA, United States of America.
authors.forename: Lynn
authors.initials: L
authors.lastname: Jackson
authors.affiliation: University of Arkansas College of Education and Health Professions, Fayetteville, AR, United States of America.
authors.forename: Donald G
authors.initials: DG
authors.lastname: Catanzaro
authors.affiliation: University of California San Diego, San Diego, CA, United States of America.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Rodwell
authors.affiliation: University of California San Diego, San Diego, CA, United States of America.
authors.forename: Antonino
authors.initials: A
authors.lastname: Catanzaro
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America; Center for Microbial Genetics & Genomics, Northern Arizona University, Flagstaff, AZ, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: Keim
authors.affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.
authors.forename: David M
authors.initials: DM
authors.lastname: Engelthaler
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology* / Q000627:therapeutic use; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D005787:Gene Frequency; D056426:Genetic Loci; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008826:Microbial Sensitivity Tests; D025202:Molecular Diagnostic Techniques; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013183:Sputum / Q000382:microbiology; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial