High-Resolution Melting Analysis for the Rapid Detection of Fluoroquinolone and Streptomycin Resistance in Mycobacterium tuberculosis

Abstract

BackgroundMolecular methods for the detection of drug-resistant tuberculosis are potentially more rapid than conventional culture-based drug susceptibility testing, facilitating the commencement of appropriate treatment for patients with drug resistant tuberculosis. We aimed to develop and evaluate high-resolution melting (HRM) assays for the detection of mutations within gyrA, rpsL, and rrs, for the determination of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis (MTB).Methodology/Principal FindingsA blinded series of DNA samples extracted from a total of 92 clinical isolates of MTB were analyzed by HRM analysis, and the results were verified using DNA sequencing. The sensitivity and specificity of the HRM assays in comparison with drug susceptibility testing were 74.1% and 100.0% for the detection of fluoroquinolone resistance, and 87.5% and 100.0% for streptomycin resistance. Five isolates with low level resistance to ofloxacin had no mutations detected in gyrA, possibly due to the action of efflux pumps, or false negativity due to mixed infections. One fluoroquinolone-resistant isolate had a mutation in a region of gyrA not encompassed by our assay. Six streptomycin-resistant strains had undetectable mutations by HRM and DNA sequencing, which may be explained by the fact that not all streptomycin-resistant isolates have mutations within rpsL and rrs, and suggesting that other targets may be involved.ConclusionThe HRM assays described here are potentially useful adjunct tests for the efficient determination of fluoroquinolone and streptomycin resistance in MTB, and could facilitate the timely administration of appropriate treatment for patients infected with drug-resistant TB.

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

“…In view of this mandate, we have developed and evaluated for the first time, to our knowledge, an HRM-based assay for direct testing of mutations responsible for RIF and INH resistance in sputum samples from a primarily M. tuberculosis SSM-negative group (84/99 samples). HRM has been proposed earlier as an alternative to conventional DST and was employed successfully as a molecular DST method for RIF, INH, and other drugs, including fluoroquinolones, streptomycin, and pyrazinamide, primarily on clinical isolates (8)(9)(10)(11)(12)(13)(14)(15)(16)19).…”

“…This method is a potentially good substitute for conventional DST in clinical practice and has been evaluated previously for the detection of RIF resistance in M. tuberculosis culture isolates from sputum, with an overall sensitivity and specificity of 94% and 99%, respectively (8). HRM has also been used successfully as a molecular DST for other drugs, including isoniazid (INH), fluoroquinolones, streptomycin, and pyrazinamide, in M. tuberculosis clinical isolates from sputum, with a sensitivity range of 74 to 100% and specificity range of 83 to 100% (9)(10)(11)(12)(13)(14)(15)(16). A microfluidic TaqMan array card that utilizes sequence-specific probes and HRM has also been reported for the detection of mutations involved in MDR and extensively drug-resistant TB (XDR-TB) in clinical isolates, with 96% accuracy (17).…”

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

“…rpsL encodes for the ribosomal protein S12 subunit, and mutations within rpsL are associated with streptomycin resistance (44). rpoB encodes for DNA-dependent RNA polymerase, and mutations within rpoB can result in rifampicin resistance (44,45).…”

“…rpoB encodes for DNA-dependent RNA polymerase, and mutations within rpoB can result in rifampicin resistance (44,45). dnaK encodes heat shock protein that is the most abundant protein within Mtb and plays a crucial role in preventing partially misfolded proteins from aggregating by binding to hydrophobic residues, which then allows these proteins to refold (46).…”

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis: a Meta-Analysis

“…HRMA is a rapid, simple, cost-effective, and closed-tube method, and it has been applied to a variety of diseases, such as inherited, infectious, and oncological diseases (14). This PCR-based method was successfully used for rapid identification and susceptibility testing of Mycobacterium tuberculosis in previous studies (2,4,15,16). On the other hand, previous studies have demonstrated that RIF resistance is a good surrogate marker for MDR-TB, especially when molecular methods are used for rapid detection of RIF-resistant M. tuberculosis isolates, as 90% of RIF-resistant M. tuberculosis isolates, or an even greater proportion, are also resistant to INH (17)(18)(19)(20).…”