Simplified Microarray System for Simultaneously Detecting Rifampin, Isoniazid, Ethambutol, and Streptomycin Resistance Markers in Mycobacterium tuberculosis

Abstract

bWe developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. M ycobacterium tuberculosis infects one-third of the world's population, with approximately nine million new cases and two million deaths attributable to the disease each year (1). Early case detection and rapid treatment are considered the most effective control strategies to reduce M. tuberculosis transmission (2), especially in cases involving multidrug-resistant (MDR) or extensively drug-resistant (XDR) M. tuberculosis. Culture-based methods remain the gold standard for diagnosing drug-resistant M. tuberculosis but can take several weeks or months to complete. Thus, nucleic acid-based drug susceptibility tests are becoming increasingly attractive as diagnostic tools in order to initiate individualized, patient-appropriate treatment in a timely manner.Technologies such as Cepheid's GeneXpert and Hain line probe assays reduce the time to diagnosis for many tuberculosis (TB) patients, provide a rapid read-out indicating resistance to rifampin or selected mutations conferring resistance to other firstor second-line drugs, and illustrate the potential to deploy molecular tests closer to the point of need (3). However, the number of known genes and mutations conferring resistance to first-and second-line drugs greatly exceeds the multiplexing capacity of these platforms, which may limit their clinical efficacy in the treatment and control of multidrug-resistant or extensively drug-resistant TB. Planar and suspension microarrays are well suited to address the multiple-gene, multiple-mutation challenge of diagnosing drug-resistant TB (4-12), but clinical adoption of microarray technology is hampered by poor reproducibility (13-15), complex workflows, and/or extensive user subjectivity and involvement in image and data analysis (16). In order to translate microarrays into efficacious TB diagnostics at the point of need, it is therefore necessary to simplify user interaction with the technology while retaining the ability to detect multiple genes and multiple mutations in a timely manner. The objectives of this study were to develop a gel element microarray test for MDR TB at a level of coverage surpassing what is currently available with WHO-endorsed molecular platforms, estimate the analytical specificity of the test on M. tuberculosis isolates of known genotype and phenotype, and compare the performance of the test to that of conventional drug susceptibility testing as a precursor to integrating the method into an entirely closed-amplicon consumable (17, 18) and sample-to-answer system. MATERIALS AND METHODSIsolates and positive control...

A Global Perspective on Pyrazinamide Resistance: Systematic Review and Meta-Analysis

“…The identification of causal PZA mutations is further complicated by the fact that not all non-synonymous mutations cause phenotypic resistance [ 80 ] and that mutations in the pncA gene can be absent in a small percentage of phenotypically PZA resistant isolates, [ 66 , 102 ] suggesting that PZA resistance could be conferred via an alternative mechanisms such as mutations in the rpsA gene [ 81 ]. Whereas development of simplified micro-array systems for simultaneous detection of rifampicin, isoniazid and ethambutol resistance may be possible, [ 131 ] inclusion of assessment of PZA resistance may thus require a different approach such as targeted DNA sequencing or next generation sequencing [ 127 , 132 ].…”

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria

“…Here, products are amplified by using mass-modified terminators detected by MALDI-TOF (130). Furthermore, microarray technology can be used to simultaneously detect a number of mutations within different genes (131,132). Multiplexing can also be achieved by a multiplex ligation-dependent probe amplification assay (MLPA) (133).…”

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

“…These technologies identify and characterize DR-TB infections based upon the presence or absence of known resistance-conferring mutations in the Mycobacterium tuberculosis genome (3)(4)(5). Unfortunately, the vast majority of rapid molecular DR-TB diagnostic tests, including line probe and microarray assays, rely on a closed set of mutations for resistance detection (6)(7)(8). The decision of which mutations to include in these assays is generally based upon the global frequencies of known resistance-associated mutations and the strength of the association between these mutations and phenotypic drug resistance to corresponding antituberculosis drugs of interest.…”