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

Abstract

BackgroundRapid 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.MethodsWe 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.ResultsThe 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%.ConclusionOur 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.

Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis Isolates from Moroccan Patients: Systematic Review

“…A total of 1589 clinical isolates were collected from TB confirmed pulmonary patients [ 24 , 26 , 28 , 29 ], and 203 isolates were collected from suspected TB patients [ 25 , 27 ]. 25.55% (458/1792) of TB patients were new cases, 30.25% (542/1792) were previously treated, and 1.84% (33/1792) of patients were under treatment.…”

“…Conventional drug susceptibility testing (DST) was performed on a Lowenstein–Jensen (L-J) medium [ 24 – 26 , 29 ] or using the proportion method [ 27 , 28 ]. Results showed various phenotypic resistance profiles ( Table 2 ); out of total 1792 MTB specimens, 874 (48.77%) were susceptible for all first-line drugs, 248 (13.84%) were isoniazid resistant (INH R ), 436 (24.33%) were RIF resistant, and 234 (13.05%) were MDR.…”

“…Results showed various phenotypic resistance profiles ( Table 2 ); out of total 1792 MTB specimens, 874 (48.77%) were susceptible for all first-line drugs, 248 (13.84%) were isoniazid resistant (INH R ), 436 (24.33%) were RIF resistant, and 234 (13.05%) were MDR. Among 918 MTB strains phenotypically resistant to rifampicin and/or isoniazid (248 INH R , 436 RIF R , and 234 MDR), 495 (238 RIF R , 234 MDR, and 23 INH R ) were subjected to rpoB mutation analysis by various genotyping resistance tests: PCR and DNA sequencing [ 25 , 26 ], qPCR-HRM [ 29 ], and rifoligotyping [ 24 ], where the genotypic tests were done after culture and the DNA was immediately used or stored at −20°C until use; however, for GenoType® MTBDR plus V2.0 [ 27 , 28 ], the assay was applied directly in the sputum specimens or after solid culture ( Table 2 ). In comparison to phenotypic data, 49 (10.38%) (8 MDR and 41 RIF R ) MTB strains phenotypically resistant to RIF did not exhibit any mutation in the rpoB gene ( Table 2 ).…”

<p>Prevalence and Molecular Typing of Colistin-Resistant <em>Pseudomonas aeruginosa</em> (CRPA) Among β-Lactamase-Producing Isolates: A Study Based on High-Resolution Melting Curve Analysis Method</p>

“…The significant advantage of the HRMA assay in this study was the identification of co-existence of CRPA and β-lactamase producing strains in a short time. Bentaleb et al 39 and Otaguiri et al 40 demonstrated that HRMA based methods have an improved level of sensitivity when compared to culture and, thus, can detect lower levels of bacteria.…”

Surveillance of Plasmodium falciparum pfcrt haplotypes in southwestern Uganda by high‐resolution melt analysis

“…In this report, a PCR-based high-resolution melt (HRM) assay was used to screen 244 clinical samples from Southwestern Uganda for pfcrt mutant haplotypes. HRM is moderate throughput and yields few false positives [29,30], exhibits high sensitivity and specificity in a variety of organisms [31][32][33], and can be employed inexpensively to screen samples prior to sequencing [30]. A previous highly accurate HRM assay of pfcrt haplotypes [34] was adopted for use on a distinct HRM instrument, evaluated for its performance with different reagents and sample types, and assessed for its ability to detect mixed alleles.…”