Adaptation of the molecular bacterial load assay for real-time quantification of <i>Mycobacterium tuberculosis</i> in the clinical routine laboratory

ABSTRACT Rapid quantification of Mycobacterium tuberculosis (Mtb) is important for monitoring bacterial load during treatment and in clinical studies. The molecular bacterial load assay detecting Mtb 16S ribosomal RNA (TB-MBLA) can quantify mycobacterial load within hours. As the current TB-MBLA protocol includes highly toxic chemicals and manual RNA extraction, we aimed to optimize TB-MBLA for routine clinical laboratories. We compared several strategies to inactivate Mtb in sputum samples, which would facilitate analysis outside a BSL3 laboratory. Heating at 80°C for 20 min was the most efficient inactivation strategy, whereas GTC-DTT and 1 M NaOH failed to inactivate Mtb sufficiently. We used Mtb-inoculated sputum samples to compare manual phenol-chloroform RNA extraction, as recommended by the TB-MBLA protocol, to an automated magnetic bead-based extraction. Automated RNA extraction reduced total analysis time by 2 h and performed equally to phenol-chloroform extraction, with a detection limit of close to 100 CFU in 10-fold dilution series (mean Ct value: 36.0 ± 1.4 automated versus 33.5 ± 3.6 manual, n = 6). Freezing samples in lysis buffer up to 14 days did not affect TB-MBLA performance (mean Ct value: 22.6 ± 0.3 fresh versus 22.6 ± 0.6 frozen, n = 6). The final TB-MBLA protocol quantified bacterial load up to 11 days faster than time-to-positive culture (TTP) (mean TTP: 11 days, 2–24 days, n = 9) in sputum from patients with pulmonary TB. We adapted the TB-MBLA method for faster throughput in a routine clinical laboratory by using inactivation at 80°C for 20 min, followed by automated RNA extraction, achieving equal sensitivity compared to the original TB-MBLA method, and avoiding manual extraction and toxic reagents. IMPORTANCE The current tuberculosis molecular bacterial load assay (TB-MBLA) protocol relies on toxic chemicals and manual RNA extraction, making the procedure both time‑consuming and labor‑intensive. We optimized and validated a faster TB-MBLA strategy, which can be adapted for routine clinical laboratories, including inactivation by heating, to facilitate implementation in settings without a BSL3 laboratory.