Abstract
Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.
Highlights
Mycobacterium tuberculosis (MTB) caused 9 million cases of tuberculosis (TB) and 1.5 million death worldwide in 2013 [1]
Our results present the first whole genome sequencing (WGS) based characterization of XDR-TB strains from Pakistan
We focused on single nucleotide polymorphisms (SNPs) in genes known to confer resistance to first- and second-line drugs and present data relevant to molecular diagnostic assays for drug susceptibility testing
Summary
Mycobacterium tuberculosis (MTB) caused 9 million cases of tuberculosis (TB) and 1.5 million death worldwide in 2013 [1]. Drug resistance in MTB increases the burden due to the disease and complicates both diagnosis and treatment. Multidrug-resistant (MDR)-TB is caused by MTB resistant to at least the two first line drugs i.e. rifampicin and isoniazid. Drug resistant (XDR)-TB is caused by MDR-TB strains resistant to fluoroquinolone and to any one of the injectable aminoglycosides; amikacin, kanamycin or capreomycin. Appropriate MDR-TB management requires drug susceptibility testing to guide treatment regimens. The usual turnaround time for phenotypic MTB drug susceptibility testing (DST) is about 8–10 weeks, often leading to delays in initiation of appropriate therapy.
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