Cross-Species Evaluation of Mycobacterium tuberculosis Complex Molecular Diagnostic Kits for Human and Veterinary Use: Introducing the One Health Perspective into Laboratory Diagnosis

Background: Molecular assays for the Mycobacterium tuberculosis complex (MTBC) are validated and authorized separately for human and veterinary use, although they target conserved sequences of the same pathogen. This separation, driven by regulatory rather than biological reasons, limits the flexible use of available diagnostics, particularly in low- and middle-income countries. We assessed whether a human-intended and a veterinary-intended MTBC real-time PCR kit retain their ability to detect MTBC in clinical samples from their non-target host. Methods: Human (n = 108; Xpert MTB/RIF Ultra-positive) and bovine (n = 100; MGIT culture-positive) clinical samples were tested with the Viasure (human) and ID Gene (veterinary) kits using the same DNA extract. We compared each kit’s sensitivity to the reference method, agreement between kits, and paired Cq values. Results: In human samples, sensitivity was 88.9% (95% CI 81.4–94.1) for ID Gene and 77.8% (68.8–85.2) for Viasure (p = 0.002). All detection failures occurred in 41 samples with longer storage and additional freeze–thaw cycles, with no evidence of PCR inhibition; the remaining 67 samples had sensitivities of 98.5% and 100%, respectively. In bovine samples, both kits showed equivalent sensitivity (74.0% [64.3–82.3] and 75.0% [65.3–83.1]; p = 1.000). Agreement between kits was 87.0% (κ = 0.54) in human and 91.0% (κ = 0.76) in bovine samples. Among samples detected by both kits, ID Gene amplified earlier than Viasure by a similar margin in human and bovine samples (median 2.5 and 2.7 cycles; p < 0.001). Conclusions: Both kits detected MTBC in samples from their non-target host, with a relative performance independent of host species; differences in sensitivity were explained by sample storage and DNA load rather than by host-related factors. These findings support a One Health approach to validating MTBC molecular diagnostics, while specificity across hosts should be confirmed with MTBC-negative samples.