Vetnews | Maart 2026 26 « BACK TO CONTENTS Article Zoonotic Tuberculosis.... <<< 25 In routine practice, conventional diagnostic methods for zTB largely mirror those used for M. tuberculosis, but they lack sufficient discriminatory power to differentiate MTBC species. These methods include the following: Smear Microscopy Smear microscopy is widely used due to its rapid turnaround time and low cost. However, its sensitivity is reduced in extrapulmonary disease, and it does not allow species-level differentiation within MTBC. Culture Culture remains the reference standard for tuberculosis diagnosis, as it enables downstream species identification and drug susceptibility testing. Nevertheless, it is limited by long turnaround times (3–8 weeks), the requirement for BSL-3 facilities, and reduced sensitivity in paucibacillary specimens. Although automated liquid culture systems shorten detection times, variable growth patterns of species such as M. bovis, M. caprae, and M. orygis may lead to misclassification or non-detection unless appropriate media modifications, such as pyruvate supplementation for M. bovis, are used (11). Biochemical Tests Historically, biochemical assays were employed to differentiate MTBC species based on metabolic characteristics. Mycobacterium bovis is negative for niacin production, nitrate reduction, and catalase activity at 68°C, and is resistant to pyrazinamide, whereas M. tuberculosis typically exhibits the opposite profile (4). More recently, detection of the MPT64 antigen—present in most MTBC members except BCG—has been incorporated into rapid immunochromatographic assays to confirm MTBC identity from culture isolates, replacing older biochemical workflows. While valuable in the past, conventional biochemical tests are labourintensive, have limited discriminatory power for closely related species, and have therefore largely been replaced by molecular techniques. Histopathology In suspected cases of extrapulmonary tuberculosis, histopathological examination of tissue biopsies may demonstrate granulomatous inflammation with caseating necrosis, findings that are characteristic but not pathognomonic of TB. Ziehl-Neelsen (ZN) staining can reveal acid-fast bacilli (AFB); however, species-level identification is not possible. Histopathology thus serves as an adjunctive tool, facilitating early presumptive diagnosis but remaining insufficient for definitive species confirmation. MOLECULAR DIAGNOSTIC METHODS Molecular diagnostics have revolutionised tuberculosis diagnosis by enabling rapid detection and speciation of members of the MTBC. These tools are particularly valuable in the context of zTB, where differentiation between M. tuberculosis and animal-adapted species such as M. bovis, M. orygis, and M. caprae is crucial for clinical management, surveillance, and epidemiological understanding. Nucleic Acid Amplification Tests: GeneXpert® and TrueNat® Platforms Widely deployed nucleic acid amplification tests (NAATs), including GeneXpert® (Cepheid, Sunnyvale, CA, USA) and TrueNat® (Molbio Diagnostics, Goa, India), enable rapid detection of MTBC DNA and rifampicin resistance from a range of clinical specimens. However, their principal limitation in the context of zTB is the inability to differentiate among MTBC species. An important advantage of the semi-closed TrueNat® platform is that the extracted DNA eluate can be retained and used for downstream species-level polymerase chain reaction (PCR) assays. This feature supports integrated diagnostic algorithms for zTB surveillance, particularly in resource-limited settings. Polymerase Chain Reaction and Real-Time PCR Polymerase chain reaction-based assays remain central to MTBC detection, targeting genomic elements such as IS6110, IS1081, 16S rRNA, mpb64, rpoB, katG, and inhA. Real-time PCR (qPCR) platforms enable rapid and quantitative detection and can be applied to both pulmonary and extrapulmonary specimens. Species differentiation is achieved using two principal molecular strategies. The first involves RD analysis, in which the presence or absence patterns of specific genomic regions (e.g., RD1, RD4, RD9, and RD12) are used to distinguish MTBC members (Table 2). The second approach relies on SNP-based typing, targeting genes such as PPE55, Rv2042c, and gyrB, which are particularly useful for differentiating M. orygis and M. caprae (1). Masanga et al. (14) demonstrated a novel target specific to animal-adapted MTBC strains; however, this marker does not allow discrimination between M. bovis and the M. bovis BCG strain. Despite its diagnostic utility, PCR-based testing has several limitations. Only a limited number of commercial kits are available for MTBC species differentiation, restricting standardisation across laboratories. In addition, open PCR systems carry a risk of contamination, highlighting the importance of appropriate laboratory infrastructure and trained personnel. These requirements are often difficult to meet in peripheral or rural laboratory settings. Nonetheless, PCR remains a practical and adaptable approach for zTB detection when integrated into referral laboratory workflows, particularly in settings using the TrueNat® platform, where extracted DNA can be repurposed for downstream speciation assays. Line Probe Assays Line probe assays, such as the GenoType® MTBC VER 1. The X system (Bruker-Hain Lifescience, GmbH, Nehren, Germany) enables rapid, culture-based identification and differentiation of MTBC species via reverse hybridisation of species-specific genetic targets. These assays can distinguish several MTBC members, including M. tuberculosis, M. bovis, M. caprae, M. africanum, M. microti, and M. canettii. In addition to speciation, LPAs can detect PZA resistance via pncA mutations and identify resistance-associated mutations related to multidrug-resistant TB, particularly in the rpoB, katG, and inhA genes.Although LPAs provide rapid and reliable speciation from culture isolates, their performance depends on the availability of pure mycobacterial cultures.
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