Vetnuus | March 2026 29 mapping (3,12). In the context of zTB, WGS enables precise discrimination among M. bovis, M. orygis, M. caprae, and other MTBC members; detection of PZA resistance by identifying mutations in the pncA gene, a critical capability for managing M. bovis infections; and high-resolution phylogenetic analysis to trace cross-species transmission and investigate outbreaks, providing crucial data for public health interventions (4). Whole genome sequencing offers the most comprehensive resolution for MTBC analysis. However, widespread implementation remains constrained by the need for BSL-3 culture facilities, sequencing platforms, skilled personnel, and bioinformatics capacity, which may limit its feasibility in routine programmatic settings. Targeted Next-Generation Sequencing Targeted next-generation sequencing (tNGS) platforms, such as Deeplex® Myc-TB (Genoscreen, Lille, France), provide a focused and efficient alternative to WGS by amplifying and sequencing specific genomic regions of interest (1). These platforms are capable of detecting mutations associated with drug resistance, identifying MTBC species and sub-lineages, and generating results more rapidly and cost-effectively than conventional WGS. As such, tNGS holds promise for expanded use in both clinical and surveillance settings, particularly where comprehensive yet resource-conscious diagnostics are needed. High-Resolution Melt Analysis and Digital PCR High-resolution melt (HRM) analysis differentiates MTBC species based on the melting profiles of amplicons. It is simple, rapid, and relatively cost-effective, but may lack sufficient discriminatory power for closely related species like M. bovis and M. caprae. Digital PCR (dPCR) provides enhanced sensitivity and quantification compared to traditional real-time PCR. Its role in zTB diagnosis remains experimental, though promising for use in extrapulmonary specimens or samples with low bacillary load. Nanopore Sequencing Nanopore sequencing platforms, such as MinION™ (Oxford Nanopore Technologies, Oxford, United Kingdom), provide portability and real-time sequencing potential. While accuracy and validation are still being optimised compared to Illumina platforms, these technologies hold promise for decentralised molecular surveillance of zTB in the future. CHALLENGES AND THE NEED FOR ONE HEALTH INTEGRATION Despite growing recognition of zTB as a public health concern, multiple diagnostic, surveillance, and policy-related challenges continue to hinder its effective detection and control. These gaps are particularly pronounced in high-burden, resource-limited settings, many of which are characterised by close human–animal interfaces and unregulated dairy or meat supply chains. Underreporting and Lack of Routine Speciation Most clinical laboratories diagnose tuberculosis using smear microscopy, NAATs, or culture, none of which routinely differentiate MTBC species (8). As a result, infections caused by M. bovis, M. orygis, and M. caprae are frequently misclassified as M. tuberculosis, particularly in extrapulmonary presentations. The lack of routine species-level identification within the MTBC has several important consequences. Misclassification of zoonotic species, such as M. bovis, distorts national TB surveillance data and hampers accurate epidemiological assessment (2). From a clinical perspective, patients infected with M. bovis may receive standard first-line regimens that include PZA, to which the organism is intrinsically resistant, resulting in suboptimal treatment outcomes. Furthermore, the inability to detect zoonotic transmission chains means that public health authorities miss critical opportunities to investigate sources of infection and implement targeted control measures. Gaps in Veterinary Surveillance Veterinary diagnostic infrastructure in many low- and middle-income countries, including India, is not equipped for molecular specieslevel testing (13). Bovine TB control programs are often inconsistent, underfunded, and vary widely by region. Key challenges include the absence of routine testing in livestock and wildlife reservoirs, poor coordination between public health and veterinary authorities, and a lack of enforceable policies for mandatory milk pasteurisation or testing in many endemic areas (10). Diagnostic Fragmentation and Lack of Intersectoral Collaboration Currently, human and animal TB cases are typically diagnosed, managed, and reported independently, with limited information exchange between sectors. This fragmentation highlights the gap between current practices and the One Health framework, which emphasises collaborative surveillance and shared responses. For instance, confirmation of M. orygis infection in a human case may not prompt investigation of local livestock or wildlife populations, even in regions where cross-species transmission is suspected. Infrastructure and Capacity Limitations The tools required for species-level diagnosis, e.g., line probe assays, real-time PCR, and WGS, are typically restricted to national or academic reference laboratories. Peripheral and district-level facilities frequently lack essential resources, including molecular platforms, trained personnel, BSL-3 containment facilities, and dedicated funding for zTB surveillance. Moreover, zTB is not yet prioritised within global TB elimination strategies, despite its implications for disease persistence, antimicrobial resistance, and vulnerable populations. Lack of Data on Emerging MTBC Species Growing evidence points to the increasing relevance of less recognised MTBC species. Mycobacterium orygis is being increasingly reported in South Asia, including in pediatric extrapulmonary TB cases and across various animal reservoirs, indicating a broader zoonotic potential than previously appreciated (3,12). Similarly, M. caprae, frequently reported in Europe, is likely underdetected in other regions due to diagnostic limitations and the absence of routine speciation in many laboratories. These observations underscore the need for expanded molecular surveillance to better understand the epidemiology and clinical significance of emerging zoonotic MTBC species. Article >>>30
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