Xpert MTB/XDR LOD for detecting FQ resistance
Timely and accurate detection of fluoroquinolone resistance is essential for the effective treatment of drug-resistant tuberculosis. Heteroresistance, the coexistence of fluoroquinolone-susceptible and resistant Mycobacterium tuberculosis subpopulations within the same sample, poses a diagnostic challenge, particularly when resistant bacilli are present at low frequencies. We assessed the analytical sensitivity of the Xpert MTB/XDR assay for detecting fluoroquinolone heteroresistance conferred by gyrA_p.Asp94Gly (high-level resistance) and gyrA_p.Ala90Val (low-level resistance). Using isogenic wild-type and mutant strains, we generated meticulously validated heteroresistant mixtures ranging from 0.5% to 60% mutant bacilli. Targeted deep sequencing (Deeplex Myc-TB) reliably quantified resistant allele frequencies >1%, verifying the intended mixture ratios. Phenotypic broth microdilution testing (EUCAST) showed levofloxacin and moxifloxacin resistance even in the lowest (0.5%) mixtures, underscoring the clinical relevance of minority variants. Xpert MTB/XDR detected gyrA_p.Ala90Val resistance at =10% (inconsistently at 5%) and gyrA_p.Asp94Gly at =30% (inconsistently at 20%), confirming mutation-dependent detection thresholds. These results illustrate both the diagnostic value and limitations of Xpert MTB/XDR in identifying clinically meaningful heteroresistance. The panel of mixtures is available as thermolysates through the BCCM/ITM collection, serving as a quality-assured resource for assay validation and development.