Real-world evaluation of the FluoroType MTBDR 2.0 assay for detection of rifampicin and isoniazid resistance-conferring mutations in Mycobacterium tuberculosis complex clinical isolates

ABSTRACT Drug-resistant tuberculosis (TB) remains a major global health challenge, particularly in high-burden settings, where rapid detection of resistance to rifampicin (RIF) and isoniazid (INH) is essential for timely treatment. The FluoroType MTBDR assay is an automated real-time PCR platform for simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and resistance to RIF and INH. This study aimed to evaluate the performance of FluoroType using clinical MTBC isolates under routine laboratory conditions in a reference setting. The assay was prospectively assessed in comparison with the GenoType MTBDR plus line probe assay (LPA) using 1,841 clinical isolates, with discordant results resolved by Sanger sequencing. For MTBC detection, FluoroType achieved 100% sensitivity (1,807/1,807), 88.2% specificity (30/34), and 99.8% overall accuracy. FluoroType demonstrated 99.9% (1,683/1,685) sensitivity in detecting wild-type isolates. For resistance detection, FluoroType showed 95.5% sensitivity for RIF (42/44) and 94.4% for INH (34/36). Among multidrug-resistant isolates, sensitivity was 85.4% (35/41). Discordances were mainly associated with heteroresistance, and uncommon or synonymous mutations. Importantly, limitations in identifying specific mutations highlight the need for cautious interpretation and, when necessary, confirmatory sequencing. Overall, these findings support FluoroType as a rapid, high-throughput, and reliable tool for MTBC detection and drug resistance profiling in reference laboratories. IMPORTANCE This study is the first to prospectively evaluate the FluoroType MTBDR assay performance using a large collection of clinical isolates under routine conditions in a high-burden tuberculosis (TB) reference laboratory. It assesses detection of Mycobacterium tuberculosis complex and resistance to rifampicin and isoniazid. Drug-resistant TB remains a major global health challenge, requiring rapid and reliable diagnostic tools. FluoroType demonstrated high accuracy and operational advantages over the line probe assay (LPA), including a streamlined workflow and reduced turnaround time for high-throughput settings. Importantly, this study highlights limitations shared by molecular assays, particularly in detecting and interpreting heteroresistance, uncommon mutations, and synonymous rpoB variants, which may lead to discrepant resistance classification across platforms. These findings underscore the need for cautious interpretation of molecular results and sequencing to resolve clinically relevant discrepancies. Overall, FluoroType is a valuable addition to TB diagnostic algorithms, but results should be interpreted in the context of assay-specific limitations.