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ABSTRACTExtensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrsgenes and the promoters of inhAand eisgenes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosisin 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

ABSTRACTResistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosisstrains is largely determined by specific mutations in the rrsgene and eisgene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosisDNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (LJ) absolute concentration, mycobacterial growth indicator tubes (MGIT), and TREK Sensititre MycoTB MIC plate (MycoTB) methods. Target amplicons were also tested for mutations by Sanger sequencing. The SMB assay correctly detected 115/116 mutant and mixed sequences and 487/487 wild-type sequences (sensitivity and specificity of 99.1 and 100%, respectively). Using the LJ method as the reference, sensitivity and specificity for AMK resistance were 92.2% and 100%, respectively, and sensitivity and specificity for KAN resistance were 87.7% and 95.6%, respectively. Mutations in the rrsgene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eispromoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eispromoter mutations with AMK resistance. Among the discordant samples with AMK and/or KAN resistance but wild-type sequence at the target genes, we discovered four new mutations in the whiB75' untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB75' UTR mutations in KAN resistance.

ABSTRACTRifampin resistance in Mycobacterium tuberculosisis largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoBgene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the M. tuberculosisRRDR by melting temperature (Tm) analysis. A three-point Tmcode is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted from 589 geographically diverse clinical M. tuberculosiscultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity, 99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using the triple Tmcode. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations when present in at least 40% of the total M. tuberculosisDNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.