Leveraging rpoB amplicon signals from Xpert MTB/RIF ultra as a diagnostic opportunity for identifying Non-tuberculous mycobacteria in TB-negative specimens

Non-tuberculous mycobacteria (NTM) present a diagnostic challenge due to clinical overlap with tuberculosis (TB). The Xpert MTB/RIF Ultra assay occasionally detects rpoB probe amplification in specimens negative for the Mycobacterium tuberculosis complex (MTBC). We evaluated whether rpoB amplification could indicate NTM infection and support diagnostic decision-making. Specimens showing rpoB probe amplification but negative for MTBC by Xpert Ultra were selected from TB Screening and Treatment Centers (TBSTC) of icddr, b between January and August 2024. Samples underwent further testing using PCR targeting rpoB and IS 6110, culture, and targeted next-generation sequencing (tNGS) to detect and identify NTM species. Among 50 specimens with rpoB amplification, rpoB PCR positivity was confirmed in all cases. Atypical mycobacterial growth was observed in 43 (86%) isolates. tNGS identified 34 isolates as NTM, with Mycobacterium gordonae (20.5%) and Mycobacterium abscessus subsp. abscessus (17.6%) being the most prevalent; 3 were MTBC, and the 6 were not detected. Receiver operating characteristic (ROC) analysis of rpoB 2 threshold cycle (Ct) values demonstrated moderate diagnostic performance (AUC 0.807) with a Ct cutoff of 32.7 yielding 82.4% sensitivity, 77.8% specificity, and a positive predictive value of 93.3%. At a cutoff value of 32.7, the sensitivity and specificity for pulmonary samples were 90% and 100%, respectively, whereas for extrapulmonary samples they were 76% and 85%, respectively. These findings indicate that rpoB 2 Ct values from the Xpert Ultra assay could provide additional diagnostic utility for identifying NTMs in MTBC-negative samples without additional costs.