A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Area of Science:

• Microbiology
• Molecular Biology
• Infectious Diseases

Background:

• Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), a Category B biological substance requiring strict laboratory safety protocols.
• Handling Mtb-positive samples necessitates containment level 3 (CL3) laboratories, posing logistical and safety challenges.
• The TB molecular bacterial load assay (TB-MBLA) quantifies Mtb load using RT-qPCR, but requires non-infectious samples.

Purpose of the Study:

• To evaluate heat inactivation as a method to render Mtb sputum samples non-infectious.
• To assess RNA preservation for TB-MBLA analysis after heat inactivation.
• To ensure worker safety during sample processing for TB diagnostics.

Main Methods:

• Sputum samples were subjected to heat inactivation at 80°C, 85°C, or 95°C for 20 minutes.
• Inactivation efficacy was confirmed by 42-day cultivation of treated and control samples.
• RNA was extracted from inactivated samples and analyzed using RT-qPCR for Mtb 16S rRNA quantification.

Main Results:

• Heat inactivation at all tested temperatures successfully rendered Mtb non-infectious, with no growth observed in cultures.
• RNA integrity was maintained, allowing for quantification via TB-MBLA.
• A minor loss of <1 log10eCFU/mL was observed due to heat-induced cell lysis.

Conclusions:

• Heat inactivation is a viable method to safely process Mtb sputum samples for TB-MBLA.
• This method enhances laboratory safety by eliminating the need for CL3 containment for sample preparation.
• Further investigation is needed to assess the impact of heat inactivation on false-negative results in low-bacterial-load TB cases.