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.