Dataset Information

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients.

ABSTRACT: Rapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.We report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates.The sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%.Our qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories.

SUBMITTER: Bentaleb EM

PROVIDER: S-EPMC5547500 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients.

Bentaleb El Mehdi EM El Messaoudi My Driss MD Abid Mohammed M Messaoudi Malika M Yetisen Ali K AK Sefrioui Hassan H Amzazi Saaïd S Ait Benhassou Hassan H

BMC infectious diseases 20170807 1

<h4>Background</h4>Rapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings.<h4>Methods</h4>We report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mu ...[more]

PMID: 28784099

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Project description:Discordant results between genotypic drug susceptibility testing (gDST) and phenotypic DST (pDST) for Mycobacterium tuberculosis isolates with disputed (discordance between gDST and pDST results) mutations affect rifampin (RIF)-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) treatments due to a lack of practical clinical guidelines. To investigate the role of disputed rpoB mutations in M. tuberculosis and TB treatment outcomes, initial isolates of 837 clinical RR- or MDR-TB cases confirmed during 2014 to 2018 were retested using agar-based RIF pDST and rpoB gene sequencing. MICs were determined for isolates with disputed rpoB mutations. Disputed rpoB mutations were identified in 77 (9.2%) M. tuberculosis isolates, including 50 (64.9%) and 14 (18.2%) phenotypically RIF- and rifabutin (RFB)-resistant isolates, respectively. The predominant single mutations were those encoding L533P (a change of L to P at position 533) (44.2%) and L511P (20.8%). Most of the isolates harboring mutations encoding L511P (87.5%), H526N (100%), D516Y (70.0%), and L533P (63.6%) had MICs of ≤1 mg/liter, whereas isolates harboring the mutation encoding H526L (75%) had a MIC of >1 mg/liter. Of the 63 cases with treatment outcomes available, 11 (17.5%) cases died, 1 (1.6%) case transferred out, and 51 (81%) cases had favorable outcomes, including 8 and 20 cases treated with standard-dose RIF- and RFB-containing regimens, respectively. Excluding cases that transferred out or received no or 1-day treatment, we observed statistically significant differences between the outcomes using active and inactive fluoroquinolones (FQs) (P = 0.008, odds ratio = 0.05 [95% confidence interval, 0.01 to 0.38]) in 57 cases (where active means a case susceptible to the drug and inactive means a case resistant to the drug or drug not used). We concluded that disputed rpoB mutations are not rare. Depending on the resources available, sequencing and/or MIC testing is recommended for better management of RR- and MDR-TB cases.

Dataset Information

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients.

Publications

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients.

Similar Datasets

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