P028

Timely detection of bedaquiline-resistant tuberculosis is critical for effective treatment of rifampicin-resistant tuberculosis, particularly in light of BPaL and BPaLM regimens. Without a rapid molecular diagnostic, bedaquiline resistance is assessed by sequencing the Rv0678 gene. Heteroresistance, the coexistence of drug-susceptible and drug-resistant populations, is reported frequently in clinical isolates and challenges the accurate diagnosis of bedaquiline resistance. We generated bedaquiline heteroresistant mixtures for inclusion in the Belgian Coordinated Collections of Microorganisms (BCCM) at 0.5%-100% resistant allele ratios.  We used an Rv0678-wild-type bedaquiline-susceptible Mycobacterium tuberculosis (Mtb) strain and its in-vitro selected isogenic daughter strain with an Rv0678 insertion mutation (192-193_ins_G), a variant reported in clinical isolates. Genotypic assessment of resistant over susceptible populations was done using Deeplex Myc-TB. Bedaquiline minimum inhibitory concentration (MIC) testing was done by the EUCAST broth microdilution method (critical concentration of 0.25 µg/ml). Deeplex accurately identified resistant populations in all mixtures ≥5%, with mutant reads detectable in the BAM files for 0.5%-3% mixtures. Mixtures at 0.5%-5% showed a bedaquiline MIC above the critical concentration, but lower than the MIC of the strain with a 100% resistant variant. Mixtures with a ≥10% resistance allele had the same bedaquiline MIC as the strain with a 100% resistant allele. These findings underscore the need for robust detection of heteroresistance, even at very low allele frequencies. This study enriches the repertoire of drug-resistant Mtb mixtures in BCCM/ITM, an important asset for research and development by facilitating the advancement of diagnostic assays for bedaquiline resistance.