Genotypic Susceptibility Testing of Mycobacterium tuberculosis Isolates for Amikacin and Kanamycin Resistance by Use of a Rapid Sloppy Molecular Beacon-Based Assay Identifies More Cases of Low-Level Drug Resistance than Phenotypic Lowenstein-Jensen Testing

Abstract

eResistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including LowensteinJensen (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 rrs gene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eis promoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eis promoter 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 whiB7 5= untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB7 5= UTR mutations in KAN resistance.T uberculosis (TB) was declared a global public emergency nearly 20 years ago (1). Although the rate of new cases of TB has been decreasing worldwide, the millennium developmental goal target of a 50% disease reduction by 2015 is unlikely to be achieved (1). An increase in the incidence of multidrug-resistant (MDR) and extensively drug resistant (XDR) TB is a serious threat to these reduction goals (1). Patients with drug-resistant TB are best identified as rapidly as possible so that appropriate infection control and treatments can be quickly initiated (2).Conventional phenotypic methods can take weeks to months to fully define the drug resistance pattern of Mycobacterium tuberculosis isolates due to the very slow growth of this bacterium (3-5). Molecular tests offer the promise of more rapid drug resistance detection. A number of tests are available to detect resistance to many of the first-line anti-TB drugs (6-8). However, there are fewer rapid tests available to test for resistance to the injectable second-line drugs amikacin (AMK), kanamycin (KAN), and capreomycin (CAP). DNA sequencing studies suggest that most cases of resistance to injectable drugs can be identified by detecting mutations in positions 1401, 1402, and 1484 in the M. tuberculosis 16S rRNA (rrs) gene and between nucleotides Ϫ...

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.…”

“…There is increasing evidence that different resistance mutations may be associated with different MICs and may confer variable susceptibilities to different drugs within a given drug category (34,35,(44)(45)(46). We investigated whether the T m generated by each SMB could be used to specifically identify individual mutations (Table 1).…”

“…We have previously described a series of real-time PCR assays that together detect most cases of resistance to FQs, AMK, and KAN in M. tuberculosis (34,42). Resistance was detected by hybridizing sloppy molecular beacons (SMBs) to amplicons of drug resistance targets and then measuring the melting temperature (T m ) of the SMB-target hybrid.…”

“…Details of the microbiological evaluation of the clinical samples are fully described in the supplemental material. For sequencing of the target genes, genomic DNA was isolated from the clinical isolates as described previously (34) and sequencing of the six target genes and promoter regions was performed as described previously (56,57). The performance of the XDR assay was evaluated against that of the MGIT drug susceptibility testing assay and sequencing as the "gold standard.…”

“…Approximately 90% of cases of INH resistance can be detected by the presence of mutations in katG codon 315 and nucleotides Ϫ8 to Ϫ15 of the inhA promoter (28); approximately 90% of cases of FQ resistance can be detected by the presence of mutations in codons 88 to 94 of the gyrA gene, with mutations in codons 500 and 538 to 540 of the gyrB gene identifying resistance in an additional few percentage of cases (29)(30)(31)(32)(33). Mutations in positions 1401 and 1402 of rrs identify approximately 80% of cases of AMK resistance, and the same rrs mutations plus mutations at positions Ϫ8 to Ϫ37 of the eis promoter identify resistance in approximately 80% of cases of KAN resistance (34)(35)(36). The presence of a wild-type sequence (or of a few rare, previously identified polymorphisms that do not encode resistance) indicates susceptibility to the same drugs with close to 100% specificity when discordant results are carefully investigated.…”