Rapid, High-Throughput Detection of Rifampin Resistance and Heteroresistance in Mycobacterium tuberculosis by Use of Sloppy Molecular Beacon Melting Temperature Coding

Abstract

M ultidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis is increasing worldwide (9, 27, 37). Rapid methods to detect drug resistance are needed to quickly identify drug-resistant strains and to implement appropriate therapy (4,16,36). M. tuberculosis does not naturally contain plasmids, and almost all cases of clinical drug resistance are caused by single-nucleotide polymorphisms (SNPs) or small insertions/deletions in relevant genes (28). In the case of rifampin resistance, 95 to 98% of rifampin-resistant clinical strains have mutations in the 80-bp rifampin resistance determining region (RRDR) of the M. tuberculosis RNA polymerase beta (rpoB) gene (9,11,12,15,20,28). PCR and probe-based molecular genotyping assays can be used to detect these resistance-inducing mutations. Such genotypic assays are potentially more rapid than labor-intensive culture-based drug susceptibility tests. Genotypic drug susceptibility testing has shown good overall concordance with the phenotypic antibiotic susceptibility tests of MDR and XDR clinical strains (6), and in a recent study, a genotypic test actually correlated better with clinical outcome than standard phenotypic susceptibility testing (36). When combined with automated sample processing systems, such as the Xpert MTB/RIF test (14), genotypic susceptibility tests can significantly reduce testing turnaround time, increasing patient notification rates and decreasing time to treatment (4, 32).The Xpert MTB/RIF assay is one example of a genotypic test that is being increasingly used to screen for rifampin resistance (33). However, the single-use cartridge design of the Xpert assay limits its use for laboratory-based high-throughput testing. Several widely used reverse blot hybridization assays, such as the INNO-LIPA Rif.TB assay (Innogenetics, Belgium) and the MTBDRplus (Hain, Germany) assay (1,5,15,19,21,23,29,34), are available for laboratory-based rifampin resistance screening; however, these assays are complicated by their open hybridization format. Open hybridization systems require a relatively cumbersome work process, including rigorous physical separation of different work areas (2, 23) due to the risk of handling open PCR amplicons in a molecular diagnostic laboratory. Open systems also require a relatively large number of probes to test for relevant resistanceassociated mutations. This requirement complicates assay chemistry and hybridization parameters. In contrast to reverse blot hy-

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

“…Multiple PCR assays will therefore be needed, and optimization of assay conditions is extremely demanding due to complex oligonucleotide interactions in a multiplex format. In contrast, a more desirable and feasible approach to distinguish between closely related genotypes is to use asymmetric PCR in conjunction with MB probe-based melting curve analysis (14,15). During asymmetric PCR, a singlestranded amplicon is produced, allowing the probe to anneal and generate fluorescence at low temperature.…”

Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing

“…This can arise during suboptimal drug treatment (acquired resistance) or due to mixed infection by strains with different susceptibilities, e.g., superinfection with a resistant strain in a patient with drugsusceptible TB (9). Chakravorty and colleagues reported that an rpoB Sanger sequencing assay required at least 50% of mutant DNA target in a background of wild-type DNA in order to be detected (10). Phenotypic DST methods commonly define a bacterial population as resistant if 1% or more of the organisms are resistant (11).…”

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use

“…The long probe length of the SMB probes and their mismatch tolerance capacity allowed us to design probes with programmable hybridization kinetics that could identify a wide range of mutations using a relatively small number of probes (34,42,53,54). Our analytic studies demonstrated robust and reproducible T m peaks that permitted error-free differentiation between wild-type and mutant target sequences, even at limiting concentrations of the target sequence.…”