Rapid detection of rifampicin, isoniazid and streptomycin resistance in Mycobacterium tuberculosis clinical isolates by high-resolution melting curve analysis

Yadav, Rakesh; Sethi, Sunil; Mewara, Abhishek; Dhatwalia, Sunil Kumar; Gupta, Dheeraj; Sharma, Mukut

doi:10.1111/j.1365-2672.2012.05379.x

Cited by 20 publications

(19 citation statements)

References 17 publications

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“…The SMB assay also avoids potential problems associated with alternative methods for mutation detection. High-resolution melt curve analysis requires the ability to detect subtle variations in melt curves (22). Other post-PCR melt-based molecular assays must detect mutations by decoding complex fluorescence contours (35).…”

Section: Discussionmentioning

confidence: 99%

“…Several other molecular techniques which detect resistance to the injectable drugs have been described recently. These include in-house reverse line blot hybridization assays, multiplex allele-specific PCR, mass spectrometry of PCR products using iPLEX Gold assay (MassArray System; Sequenom, Inc., San Diego, CA, USA), oligonucleotide microarray, and melting curves using dually labeled probes (19)(20)(21)(22)(23)(24). However, most of these techniques require open handling of PCR amplicons or have not been systematically validated in clinical settings.…”

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confidence: 99%

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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

et al. 2015

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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...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

et al. 2015

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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

et al. 2017

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Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INHinhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% ( value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR] 400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.KEYWORDS DNA sequencing, high-resolution melt curve analysis, tuberculosis, molecular diagnostics, multidrug resistance, sputum T uberculosis (TB) is one of the world's deadliest transmittable diseases (1). In 2015, worldwide, 10.4 million people were estimated to be infected with TB, with 1.4 million people dying from the disease. Drug-resistant TB is a menace for TB control worldwide, and 60% of reported TB patients estimated to have multidrug-resistant TB (MDR-TB) were not detected in 2015 (1). India holds the dubious distinction of harboring 27% of the global TB burden, with 2.5% of its new TB cases and 16% of previously treated cases having MDR-TB (1). Conventional Mycobacterium tuberculosis culturebased systems for first-line and second-line drug susceptibility testing (DST) are timeconsuming, while commercial liquid culture-based DST reduces turnaround times but

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“…In 2010, 8.8 million new cases of TB and 1.4 million TB-related deaths were reported worldwide. The emergence of multidrugresistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) and even totally drug-resistant TB (TDR-TB) has significantly threatened TB control efforts, although the global incidence of TB appears to be decreasing (1,2). MDR-TB is defined as TB that is resistant to both isoniazid (INH) and rifampin (RIF), with or without resistance to other anti-TB drugs.…”

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

Yin

Liu

et al. 2013

J Clin Microbiol

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A rapid, simple, accurate, and affordable method for the detection of drug-resistant tuberculosis is very critical for the selection of antimicrobial therapy and management of patient treatment. High-resolution melting curve analysis has been used for the detection of rifampin resistance in Mycobacterium tuberculosis and has shown promise. We did a systematic review and metaanalysis of published studies to evaluate the accuracy of high-resolution melting curve analysis for the detection of rifampin resistance in clinical M. tuberculosis isolates. We searched the PubMed, BIOSIS Previews, and Web of Science databases to identify studies and included them according to predetermined criteria. We used the DerSimonian-Laird random-effects model to calculate pooled measures and applied Moses' constant for linear models to fit the summary receiver operating characteristic curve. According to the selection criteria, most of the identified studies were excluded, and only seven studies were included in the final analysis. The overall sensitivity of the high-resolution melting curve analysis was 94% (95% confidence interval [CI], 92% to 96%), and the overall specificity was very high at 99% (95% CI, 98% to 100%). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 63.39 (95% CI, 30.21 to 133.00), 0.06 (95% CI, 0.04 to 0.09), and 892.70 (95% CI, 385.50 to 2,067.24), respectively. There was no significant heterogeneity across all included studies for the measurements we evaluated. The summary receiver operating characteristic curve for the same data shows an area of 0.99 and a Q* value of 0.97. High-resolution melting curve analysis has high sensitivity and specificity for the detection of rifampin resistance in clinical M. tuberculosis isolates. This method might be a good alternative to conventional drug susceptibility tests in clinical practice.

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Rapid detection of rifampicin, isoniazid and streptomycin resistance in Mycobacterium tuberculosis clinical isolates by high-resolution melting curve analysis

Cited by 20 publications

References 17 publications

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

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

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

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

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