## 4 The Use of Mycobacteriophages in Diagnostics and Therapy
### Phage-Based Diagnostic Strategies for Mycobacteria  
In response to these diagnostic limitations, phage-based diagnostic approaches are gaining traction [14]. Since the first identification of mycobacteria in 1947, researchers have explored their use in detection, species identification, and drug susceptibility testing for TB [40; 95]. These assays offer several advantages: rapid turnaround, minimal instrumentation, and low operating costs. Two primary phage-based diagnostic strategies are currently in use: Phage Reporter Assays (PRA) and Phage Amplification Assays (PAA) [96]. Both methods rely on viable mycobacterial cells to support phage replication or reporter gene expression. The PRA uses genetically modified mycobacteria that carry reporter genes such as luciferase (Fflux or luc) or fluorescent proteins like GFP or ZsYellow [51; 96]. Whena modified phage infects a viable host cell, the reporter gene is expressed, resulting in bioluminescence (measurable by luminometer) or fluorescence (detectable by microscopy or flow cytometry), depending on the gene used (Figure 3). The presence of a light emission indicates metabolically active bacteria in the sample [51; 96]. Although PRA is sensitive and fast, its specificity may be limited, as some phages--such as TM4--can infect multiple _Mycobacterium_ species (_M. tuberculosis_, _M. bovis_, and _M. avium-intracellular_) [96]. In culture-based differentiation of mycobacterial species, the chemical inhibitor p-nitro-\(\alpha\)-acetylamino-\(\beta\)-hydroxyl-propiophenone (NAP) is employed to enhance specificity by selectively inhibiting the growth of _M. tuberculosis_ complex strains, which facilitates their distinction from non-tuberculous mycobacteria that may otherwise interfere with diagnostic accuracy [51; 97]. Parallel tests with and without NAP allow species differentiation based on the presence or absence of a luminescent signal. Other mycobacteriophages utilized in these assays include L5, Che12, D29, and the highly specific DS-6A [96; 97]. In drug susceptibility testing, fluorescent phage-based tools offer a real-time assessment of bacterial response to antimicrobials, further enhancing the clinical value of phage diagnostics [98; 99; 100]. [PERSON] et al. demonstrated that tests based on luciferase reporter phages (LRP) exhibit comparable sensitivity, specificity, and rapidity to the MGIT 960 and BACTEC 460 systems for the detection of antibiotic-resistant M. tuberculosis in Mexico [100]. Furthermore, the LRP assay is up to five times faster than the BACTEC 460 method. Following these findings, luciferase phage-based diagnostics could be implemented in reference laboratories for mycobacterial identification in developing countries, potentially enhancing access to reliable testing [100]. The second test, PAA, uses non-recombinant phases to indirectly detect the presence of mycobacteria through morphological changes (bal  
Figure 3: Phage reporter assay. This method utilizes genetically engineered mycobacteria as vectors to deliver reporter genes into mycobacterial cells. Reporter genes such as firefly luciferase (FFLuv), green fluorescent protein (GFP), or ZsYellow are inserted into non-essential regions of the phage genome without disrupting its infectivity. Upon infection, the reporter genes are expressed by metabolically active target bacterial cells. In the case of luciferase, light emission occurs only in viable cells that possess sufficient levels of intracellular ATP and are supplied with the exogenous substrate luciferin. Thus, the bioluminescent signal is a reflection of host metabolic activity, not of the phage genome structure itself. The light output can be quantified using a luminometer or detected semi-quantitatively with a low-cost photographic film setup (e.g., Bronx Box). Fluorescent proteins such as GFP and ZsYellow accumulate in live cells and can be visualized using fluorescence microscopy or quantified via flow cytometry. Based on Refs. [51; 96; 98; 99]. lawn [51]. This test most commonly employs the D29 phage, capable of infecting _M.