Mycobacteriophages: An Integrated Platform for Rapid Diagnosis and Targeted Therapy of Tuberculosis Infection

The global spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) demands fundamentally new approaches combining diagnostic speed and therapeutic efficacy. Within the strategy of personalised medicine, the lytic mycobacteriophage D29 represents a promising tool for both phenotypic drug susceptibility testing and the targeted delivery of therapeutic agents to the infection site. We developed a two-component platform based on D29: (1) a diagnostic test system combining a rapid anti-mycobacterial susceptibility testing (AMST) module, which detects phage D29 replication in drug-resistant isolates via multiplex real-time PCR; and (2) a liposomal therapeutic formulation. Therapeutic efficacy was evaluated in C57BL/6 mice (n = 55) with experimental pulmonary tuberculosis receiving liposomal D29 either aerosolised (inhalation chamber) or intranasally. Bacterial burden (CFU/lung) was assessed at days 3 and 14 post-therapy. Pharmacokinetics were studied in 90 mice using the double-layer agar method. The diagnostic test showed an average sensitivity and specificity of 90.0% and 96.3% across nine drugs, with results available within five days. The lyophilised components and phage remained stable for 12 months. Aerosol therapy demonstrated a trend towards a higher efficacy compared to intranasal administration: the median CFU/lung at day 3 was 107 (i.e., one hundred and seven) vs. 23,500 (control 65,000); 60% of aerosol-treated animals achieved ≤200 CFU/lung (not statistically significant, p = 0.089). Both routes significantly outperformed controls (p < 0.01 and p < 0.05). Pharmacokinetics revealed exclusive lung tropism with peak concentrations at 5 min (1.9 × 103–5.1 × 104 PFU/organ) and phage levels falling below the limit of detection (<5 × 103 PFU/organ) within 24 h. Infected lungs showed significantly higher phage titres than uninfected ones, indicating in vivo replication. Safety studies showed no significant toxicity, immunogenicity, or local irritation. The integrated D29-based platform represents a comprehensive, integrated diagnostic–therapeutic solution covering the full “diagnosis–therapy” cycle. The test system provides rapid phenotypic drug susceptibility profiling, while the liposomal phage demonstrates potent therapeutic activity and favourable pharmacokinetics, supporting its clinical translation.