Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis

Evans, Joanna C.; Segal, Heidi

doi:10.1093/jac/dkq047

Cited by 37 publications

(24 citation statements)

References 7 publications

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“…AMK, KAN, and CAP are key drugs for treating MDR TB, and the most common mutations associated with a resistance of these drugs are located within the rrs gene coding for 16S rRNA (4,19,20). We therefore analyzed a 481-bp region of this gene.…”

Section: Resultsmentioning

confidence: 99%

Molecular Characterization of Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Jiangxi, China

et al. 2012

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In this study, a total of 77 multidrug-resistant and extensively drug-resistant (MDR and XDR, respectively) isolates of Mycobacterium tuberculosis were characterized among samples from patients living in Jiangxi province, China. The following two approaches were used: (i) genotyping all drug-resistant isolates by the 15-locus MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) method and identifying the Beijing family genotype using the RD105 deletion targeted multiplex PCR and (ii) determining the mutation profiles associated with the resistance to the first-line antituberculous drugs rifampin (RIF) and isoniazid (INH) and the second-line drugs ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) with DNA sequencing. Six loci were examined: rpoB (for resistance to RIF), katG and mabA-inhA (INH), gyrA and gyrB (OFX), and rrs (KAN, AMK, and CAP). It is shown that the Beijing genotype was predominant (80.5%) among these strains and that the selected drug-resistant strains were genetically diverse, suggesting that they probably had independently acquired drug resistance. In comparison to the phenotypic data, the sensitivities for the detection of RIF, INH, OFX, and KAN/AMK/CAP resistance by DNA sequencing were 94.8, 80.5, 84.6, and 78.9%, respectively. The most prevalent mutations involved in RIF, INH, OFX, and KAN/AMK/CAP resistance were Ser531Leu in rpoB (44.2%), Ser315Thr in katG (55.8%) and C-15T in mabA-inhA (11.7%), Asp94Gly in gyrA (48.7%), and A1401G in rrs (73.7%), respectively. Five novel katG mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro, and Asp695Gly) and six new alleles (Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu, and Lys972Thr) in the rpoB gene were identified.

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Section: Resultsmentioning

confidence: 99%

Molecular Characterization of Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Jiangxi, China

et al. 2012

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“…Rapid molecular tests that detect the rrs A1401G mutation are currently used to predict cross-resistance patterns of the injectable antibiotics and aid in the classification of patients as either pre-XDR or XDR (3,19,26,27). However, a major setback to adopting A1401G as a molecular marker of resistance is the wide range of CAP MICs observed in isolates with this rrs mutation.…”

Section: Discussionmentioning

confidence: 99%

“…Numerous reports of discordance between the genotypic results (i.e., A1401G mutation) and phenotypic CAP results call into question the validity of the A1401G mutation as an accurate predictor of CAP resistance (3,14,15,29). However, the A1401G mutation does appear to be a good predictor for KAN and AMK cross-resistance (8,14,18,26,27). This molecular marker is currently used in at least one commercially available molecular test (4), and additional rapid diagnostic assays using this mutation are being developed (4,19,27).…”

Section: Discussionmentioning

confidence: 99%

Disparities in Capreomycin Resistance Levels Associated with the rrs A1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

Reeves

Campbell

Willby

et al. 2015

Antimicrob Agents Chemother

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bAs the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for secondline drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring the rrs A1401G mutation and found that the CAP MICs ranged from 8 g/ml to 40 g/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 g/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 g/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome. Global tuberculosis (TB) control efforts are severely complicated by the emergence and spread of multidrug-resistant (MDR) TB, as evidenced by the half million new MDR TB cases reported by the World Health Organization (WHO) last year (1). MDR TB is defined as Mycobacterium tuberculosis resistant to two of the most potent first-line antituberculosis agents, rifampin (RIF) and isoniazid (INH) (1). Treatment requires the administration of second-line antibiotics that are more expensive, can lead to more adverse side effects, and increase treatment duration time relative to first-line drugs (1, 2). Unfortunately, inadequate treatment of MDR TB and the misuse of second-line antibiotics have contributed to the emergence of extensively drug-resistant (XDR) TB strains, defined by WHO as multidrug resistant with additional resistance to any fluoroquinolone and at least one of the injectable aminoglycoside agents kanamycin (KAN), amikacin (AMK), or capreomycin (CAP) (1).Rapid and reliable diagnosis of antibiotic resistance is critical in controlling the spread of drug-resistant TB. Laboratories are increasingly adopting rapid molecular methods to identify mutations associated with resistance as a complement to the more time-consuming growth-based methods (3, 4). Though molecular methods can increase specificity and drastically reduce delay in th...

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“…These differences could be due to alternate mutations in regions other than the currently known target regions for these drugs (17). In addition, mycobacterial cultures may also comprise a mixed population of resistant and sensitive bacteria (8), and therefore, DNA extracted from sample specimens may be a lower-level representation of the resistant genotype, leading to a falsely low detection rate in the specimen.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Mutations Conferring Extensive Drug Resistance to Mycobacterium tuberculosis Isolates in Pakistan

Ali

Hasan

Jabeen

et al. 2011

Antimicrob Agents Chemother

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The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistanceconferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.

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Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis

Cited by 37 publications

References 7 publications

Molecular Characterization of Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Jiangxi, China

Molecular Characterization of Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Jiangxi, China

Disparities in Capreomycin Resistance Levels Associated with the rrs A1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

Characterization of Mutations Conferring Extensive Drug Resistance to Mycobacterium tuberculosis Isolates in Pakistan

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