Abstract
Amplicon-based Next Generation Sequencing (NGS) is an emerging method for Mycobacterium tuberculosis drug susceptibility testing (DST) but has not been well described. We examined 158 clinical multidrug-resistant M. tuberculosis isolates via NGS of 11 resistance-associated gene regions covering 3519 nucleotides. Across these gene regions, complete resistance or heteroresistance (defined as 1%-99% mutation) was present in at least one isolate in 6.3% of loci. The number of isolates with heteroresistance was highest for gyrA codon 94, rpoB codons 526 and 531, and embB codons 306, 372 and 406 (range 11–26% of isolates exhibited heteroresistance). 57% of MDR strains had heteroresistance of one or more recognized resistance-associated mutation. Heteroresistant loci generally exhibited high or low degrees of mutation (>90% or <10%). The deep sensitivity of NGS for detecting low level pncA heteroresistance appeared to improve genotypic-phenotypic PZA susceptibility correlations over that of Sanger. NGS demonstrates that heteroresistance in TB in the regions of key genes is common and will need to be bioinformatically managed. The clinical significance of such heteroresistance is unclear, and further study of pncA should be pursued.
Highlights
Tuberculosis (TB) continues to be a major global health threat with an estimated 9.6 million new cases and 1.5 million deaths in 2014 alone [1]
Optimal MDR-TB therapy is guided by individualized drug susceptibility testing (DST) [2] traditional phenotypic culture-based methods are slow
The remainder were other mutations of unclear significance. Not surprisingly in these MDR-TB strains, a large number of isolates had mutation at katG 315 and the common rpoB 531 mutations, and the pncA mutations were scattered throughout the gene
Summary
Tuberculosis (TB) continues to be a major global health threat with an estimated 9.6 million new cases and 1.5 million deaths in 2014 alone [1]. Multidrug-resistant (MDR)-TB is resistant to both rifampin and isoniazid, is complicated to manage, and has poor treatment outcomes. Optimal MDR-TB therapy is guided by individualized drug susceptibility testing (DST) [2] traditional phenotypic culture-based methods are slow.
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