Genome sequencing reveals novel deletions associated with secondary resistance to pyrazinamide in MDR Mycobacterium tuberculosis

Objectives: Detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates presents significant challenges in settings with no dominant clonal lineages, such as Australia. We assessed the utility of WGS versus standard PCR amplification assays for the characterization of pyrazinamide resistance in MDR-TB isolates identified in New South Wales, Australia, over an 8 year period. Methods: PCR amplicon sequencing was used to identify molecular markers associated with antibiotic resistance in pyrazinamide-resistant MDR-TB isolates recovered by the New South Wales Mycobacterium Reference Laboratory between 2007 and 2014. WGS was subsequently performed on two isolates for which pncA amplification failed. Results: WGS identified two novel genomic deletions associated with in vitro resistance to pyrazinamide in MDR-TB. One isolate also carried a second deletion involving the genes dfrA and thyA associated with resistance to para-aminosalicylic acid. Conclusions: Steadily decreasing sequencing costs are increasing the appeal of WGS as an alternative approach for detecting complex patterns of pyrazinamide resistance in MDR-TB.