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

Ann S G Lee,Joshua C L Wong,Gilman K H Siu,Danny C T Ong,Wing-Cheong Yam,Igor Mokrousov

doi:10.1371/journal.pone.0031934

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.

Highlights

Mycobacterium tuberculosis (MTB) is the world’s leading cause of mortality due to an infectious agent with global estimates of 2 billion people currently infected worldwide [1]
The high-resolution melting (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
We aimed to develop a HRM assay to scan for mutations in gyrA, rpsL and rrs, for the determination of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis

Summary

Introduction

Mycobacterium tuberculosis (MTB) is the world’s leading cause of mortality due to an infectious agent with global estimates of 2 billion people currently infected worldwide [1]. The WHO Report 2010 on ‘‘Global Tuberculosis Control’’ documents that in 2009 there were 9.4 million incident cases of tuberculosis and approximately 1.3 million deaths (http://www.who.int/tb/publications/global_ report/2010/gtbr10_main.pdf). Complicating this scenario is the emergence of multidrug-resistant tuberculosis (MDR-TB), defined as resistance to two first-line antitubercular drugs, isoniazid and rifampin, causing great global concern and resulting in an increased need for the understanding of the molecular mechanisms and molecular epidemiology of drug resistance [2,3,4]. MDR-TB treatment requires use of second-line drugs such as fluoroquinolones (Moxifloxacin, gatifloxacin, levofloxacin) or injectable agents such as aminoglycosides (streptomycin, amikacin, kanamycin) and polypeptides (capreomycin) for typically two years [5,6]. 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)

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