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The rapid and robust identification of mutations in Mycobacterium tuberculosis complex (MTBC) strains mediating multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes is crucial to combating the MDR tuberculosis (TB) epidemic. Currently available molecular anti-TB drug susceptibility tests either are restricted to a single target or drug (i.e., the Xpert MTB/RIF test) or present a risk of cross-contamination due to the design limitations of the open platform (i.e., line probe assays). With a good understanding of the technical and commercial boundaries, we designed a test cartridge based on an oligonucleotide array into which dried reagents are introduced and which has the ability to identify MTBC strains resistant to isoniazid, rifampin, and the fluoroquinolones. The melting curve assay interrogates 43 different mutations in the rifampin resistance-determining region (RRDR) of rpoB , rpoB codon 572, katG codon 315, the inhA promoter region, and the quinolone resistance-determining region (QRDR) of gyrA in a closed cartridge system within 90 min. Assay performance was evaluated with 265 clinical MTBC isolates, including MDR/XDR, non-MDR, and fully susceptible isolates, from a drug resistance survey performed in Swaziland in 2009 and 2010. In 99.5% of the cases, the results were consistent with data previously acquired utilizing Sanger sequencing. The assay, which uses a closed cartridge system in combination with a battery-powered Alere q analyzer and which has the potential to extend the current gene target panel, could serve as a rapid and robust point-of-care test in settings lacking a comprehensive molecular laboratory infrastructure to differentiate TB patients infected with MDR and non-MDR strains and to assist clinicians with their early treatment decisions.

Rapid Microarray-Based Detection of Rifampin, Isoniazid, and Fluoroquinolone Resistance in Mycobacterium tuberculosis by Use of a Single Cartridge