title: Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study.
abstract: Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA rcolman@ucsd.edu.
authors.forename: Rebecca E
authors.identifier: 0000-0002-6854-2100
authors.initials: RE
authors.lastname: Colman
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Anderson
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: Darrin
authors.initials: D
authors.lastname: Lemmer
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Lehmkuhl
authors.affiliation: Division of Pulmonary, Critical Care, and Sleep Medicine, University of San Diego, San Diego, California, USA.
authors.forename: Sophia B
authors.initials: SB
authors.lastname: Georghiou
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Heaton
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: Kristin
authors.initials: K
authors.lastname: Wiggins
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Gillece
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Schupp
authors.affiliation: College of Education and Health Professions, University of Arkansas, Fayetteville, Arkansas, USA.
authors.forename: Donald G
authors.initials: DG
authors.lastname: Catanzaro
authors.affiliation: Phthisiopneumology Institute (PPI), Chisinau, Republic of Moldova.
authors.forename: Valeriu
authors.initials: V
authors.lastname: Crudu
authors.affiliation: Yale School of Public Health, New Haven, Connecticut, USA.
authors.forename: Ted
authors.initials: T
authors.lastname: Cohen
authors.affiliation: Division of Pulmonary, Critical Care, and Sleep Medicine, University of San Diego, San Diego, California, USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Rodwell
authors.affiliation: Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Engelthaler
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D004364:Pharmaceutical Preparations; D010865:Pilot Projects; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D055815:Young Adult
chemical_list: D004364:Pharmaceutical Preparations