title: Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a single multiplex PCR and DNA sequencing.
abstract: Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.
authors.affiliation: Washington State Public Health Laboratories, Shoreline, Washington, USA.
authors.forename: Ailyn C
authors.initials: AC
authors.lastname: Pérez-Osorio
authors.forename: David S
authors.initials: DS
authors.lastname: Boyle
authors.forename: Zachary K
authors.initials: ZK
authors.lastname: Ingham
authors.forename: Alla
authors.initials: A
authors.lastname: Ostash
authors.forename: Romesh K
authors.initials: RK
authors.lastname: Gautom
authors.forename: Craig
authors.initials: C
authors.lastname: Colombel
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Houze
authors.forename: Brandon T
authors.initials: BT
authors.lastname: Leader
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S