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

Abstract

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.

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

“…Compared with sequencing, the rpoB HRM assay had a diagnostic sensitivity of 90.5% (95% CI, 65, 99%), with 100% specificity (95% CI, 96, 100%). The sensitivity was slightly lower than that reported for M. tuberculosis culture isolates in a meta-analysis for the detection of RIF resistance (pooled sensitivity and specificity estimates of 94% and 99%, respectively [8]). This might be due to the fact that DNA was isolated directly from sputum samples with a predominantly SSM-negative (84/99 samples) sample set.…”

“…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).…”

“…Sequence modifications are demarcated by differences in the melting curve profiles among sample and reference DNA and are dependent on the base composition, length, GC content, and strand complementarity of the amplicon (7). 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).…”

Classical and New Assays for Detecting Drug Resistance in Tuberculosis

“…This capacity that took months to complete not long ago, can now be performed in less than a day for a bacterial genome [55,56]. In TB, whole-genome sequencing is being increasingly explored as a tool to study more in detail the molecular epidemiology of the disease and elucidate the origins of TB outbreaks and evolution of the TB bacillus [57,58].…”

High-resolution melt curve analysis for rapid detection of rifampicin resistance inmycobacterium tuberculosis: A single-center study in Iran

“…High resolution melting (HRM) curve analysis is a simple and robust method for detecting drug-resistant TB [16–21]. The advantages of HRM include rapid turn-around times, a closed-tube method that greatly reduces contamination risk and, unlike other methods, requires no post-PCR handling [22, 23]. The method is based on the analysis of fluorescence curves produced by DNA-binding dye during strand dissociation events in the melting phase following real-time PCR [23–25].…”