Reuse of Residual DNA Eluates from TrueNat™ Testing for Mycobacterium tuberculosis Complex Subspecies Differentiation: A Proof-of-concept Implementation Feasibility Study

BACKGROUND: The Mycobacterium tuberculosis complex (MTBC) includes human-and animal-adapted species. Routine TrueNat™ testing detects MTBC but does not differentiate subspecies. This study evaluated the reuse of residual TrueNat™ DNA eluates for MTBC subspecies identification. METHODS: A cross-sectional laboratory feasibility study was performed using residual DNA (~78 µL) from 1,115 high-bacillary-load TrueNat™ MTBC-positive clinical specimens collected between 2022 and 2025. A two-step real-time polymerase chain reaction (PCR) assay targeting IS1081, MTCHum, MTCAni, and species-specific loci for Mycobacterium bovis (Mbov), Mycobacterium caprae (Mcap), and Mycobacterium orygis (Morg) was performed. All Mbov detections underwent confirmatory PCR and culture. RESULTS: All eluates produced valid amplification. Of 1,115 samples, 1,114 (99.91%) were identified as M. tuberculosis sensu stricto and one (0.09%) as Mbov. The Mbov isolate, obtained from a 2-year-old child with lymphadenitis, was confirmed to be a Bacillus Calmette-Guérin (BCG) vaccine strain rather than wild-type Mbov. No Mcap or Morg were detected. Negative controls remained negative, and repeat testing showed 100% concordance. CONCLUSIONS: Residual DNA from routine TrueNat™ testing can be effectively reused for MTBC subspecies differentiation. This implementation feasibility study demonstrates that residual DNA from routine TrueNat™ testing can be reused for MTBC subspecies differentiation within existing workflows without additional specimen collection or DNA extraction. Dedicated validation and epidemiologic studies are required to evaluate its utility in surveillance applications.