Diagnostic luciferase reporter phage assay for active and non-replicating persistors to detect tubercle bacilli from sputum samples

Diagnosis of latent tuberculosis infection is a myth for want of a simple, direct tool. Simulation of hypoxic environment was done to create a novel hypothetical model for persistence using processed sputum samples. The adaptation of tubercle bacilli to hypoxic environment seems to be influenced by pre-existing clinical status of the patients at the time of sputum collection, resulting in varied growth pattern. Bacilli from 36 samples did not get adapted to latency of which 15 samples were from patients in whom the disease was well established and the tubercle bacilli in them probably did not experience any stress whatsoever. Similarly, 10 of the 37 samples showing the presence of cultivable cells in both aerobic and anaerobic conditions were from patients who had relapsed. The bacilli in these samples had been probably experiencing stress and thus were ready to adapt to the hypoxic environment. Diagnostic luciferase reporter phage assay for non-replicating persistors (DLRPA-NRP) identified 30 additional positives which failed to grow on Lowenstein–Jensen medium. Presence of viable bacilli in these samples was confirmed by reverse transcriptase-PCR (RT-PCR) for 16S rRNA indicating either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistors. The utility of LRP assay to detect both dormant and active tubercle bacilli was explored in this work and was optimized using lysis inhibition to diagnose tuberculosis with rapidity, improved sensitivity and specificity. DLRPA-NRP, a rapid growth based assay is thus developed to detect both dormant and actively growing tubercle bacilli.