A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

Abstract

Background: Mycobacterium tuberculosis is a category B infectious pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bacterial Load Assay (TB-MBLA).

Method: We performed in vitro experiments using M.tb, H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x10¹ CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 min and with GTC for 15 min. Inactivated samples were processed for culture and TB-MBLA.

Results: No M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log₁₀ (P = 0.7) and 1.8log₁₀ (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log₁₀ compared to 5.4 ± 3.1 log₁₀ eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative.

Conclusion: Heat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.