Rapid detection of rifampin resistance in <i>Mycobacterium tuberculosis</i> using nucleotide MALDI-TOF MS: a comparative study with phenotypic drug susceptibility testing and DNA sequencing

Rifampin (RIF) resistance in <i>Mycobacterium tuberculosis</i> (<i>M.tb</i>) is primarily caused by mutations in the <i>rpoB</i> gene. Rapid and accurate detection of RIF resistance is critical for effective tuberculosis (TB) control. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technology used to detect RIF resistance-associated <i>rpoB</i> mutations in 210 <i>M</i>.<i>tb</i> clinical isolates, including 107 RIF-sensitive and 103 RIF-resistant strains, as determined by phenotypic drug susceptibility testing (DST). DNA sequencing was used as the reference method to validate nucleotide MALDI-TOF MS results. Nucleotide MALDI-TOF MS demonstrated a sensitivity of 93.2%, specificity of 98.1%, and an overall accuracy of 95.7% compared to phenotypic DST. The Kappa value between nucleotide MALDI-TOF MS and phenotypic DST was 0.91, indicating excellent agreement. DNA sequencing confirmed that nucleotide MALDI-TOF MS successfully identified RIF resistance-associated mutations, particularly in codons 450, 445, and 435 of the <i>rpoB</i> gene. Among the 61 isolates analyzed by DNA sequencing, nucleotide MALDI-TOF MS and sequencing results were consistent for 52 of 56 RIF-resistant strains and all five RIF-sensitive strains, with an overall concordance of 93.4%. Importantly, nucleotide MALDI-TOF MS accurately detected heteroresistance in eight isolates (14.3%), confirmed by sequencing. These results support that nucleotide MALDI-TOF MS is a rapid, accurate, and reliable method for detecting <i>rpoB</i> mutations associated with RIF resistance in <i>M.tb</i>. Its high concordance with DNA sequencing, excellent diagnostic performance, and ability to identify heteroresistance highlight its potential as a valuable tool for early TB diagnostics and improve the precision of chemotherapy regimen development.IMPORTANCEThe emergence of multidrug-resistant tuberculosis (MDR-TB) and rifampin-resistant tuberculosis (RR-TB) poses a significant challenge to global tuberculosis (TB) control efforts. Rifampin (RIF) resistance is a critical marker for MDR-TB, which requires more complex, prolonged, and costly treatment regimens. Early and accurate detection of RIF resistance is crucial for effective TB control. This study evaluates the performance of nucleotide MALDI-TOF MS, an innovative technology, for detecting RIF resistance-associated mutations in the rpoB gene. The method demonstrates high sensitivity (93.2%) and specificity (98.1%), with the added advantage of identifying heteroresistance, capabilities that are lacking in conventional methods. These capabilities are crucial for early diagnosis, guiding personalized treatment regimens, and curbing the transmission of drug-resistant TB. The findings demonstrate that nucleotide MALDI-TOF MS provides a rapid, high-throughput, and cost-effective alternative for detecting rpoB gene mutations associated with RIF resistance.