Validation of novel Mycobacterium tuberculosis isoniazid resistance mutations not detectable by routine molecular tests

Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. WGS minimized the chances of missing mutations outside commonly targeted resistance hotspots, providing a higher level of confidence in unvalidated mutations as probable INH resistance determinants than was previously possible with conventional means. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. These functional genetics experiments confirm, for the first time, seven suspected INH resistance mutations and five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of ≥0.25 µg/mL and should be added to the list of high confidence INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R TB compared to current rapid molecular testing methods and could provide earlier diagnosis of drug-resistant TB.