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J Clin Microbiol. 2019 Mar 28;57(4). pii: e01778-18. doi: 10.1128/JCM.01778-18. Print 2019 Apr.

Heat Inactivation Renders Sputum Safe and Preserves Mycobacterium tuberculosis RNA for Downstream Molecular Tests.

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School of Medicine, University of St Andrews, St Andrews, United Kingdom
Instituto Nacional de Saúde (INS), Ministério da Saúde, Maputo, Mozambique.
Division of Infectious Diseases and Tropical Medicine, University of Munich, Munich, Germany.
German Centre for Infection Research (DZIF), Partner Site Munich, Munich, Germany.
School of Medicine, University of St Andrews, St Andrews, United Kingdom.


The World Health Organization End Tuberculosis (TB) strategy has called for the development of-and increased access to-effective tools for diagnosis and treatment of TB disease. Mycobacterium tuberculosis , the causative agent of TB, is categorized as a highly infectious agent. Consequently, diagnostic tests that involve comprehensive manipulation of specimens from presumed tuberculosis cases must be performed in a category 3 laboratory. We have evaluated the use of heat inactivation to render TB samples safe to work with while preserving RNA for downstream molecular tests. Using Mycobacterium bovis bacillus Calmette-Guérin (BCG) cultures and TB-positive sputum samples, we show that boiling for 20 min at 80, 85, and 95°C inactivates all M. tuberculosis bacilli. The efficiency of inactivation was verified by culturing heat-treated and untreated (live) fractions of BCG and TB sputum samples for 42 days. No growth was observed in the cultures of heat-treated samples. In contrast, the optical density of untreated BCG in Middlebrook 7H9 broth rose from 0.04 to 0.85, and the untreated sputum samples flagged positive at 3 days of incubation in mycobacterial growth indicator tubes. Quantification of reference genes 16S rRNA, transfer-messenger RNA (tmRNA), pre-16S rRNA, and rpoB by reverse transcriptase quantitative PCR (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA species dependent, <1 log10, 1.1 log10, 1.3 log10, and 2.4 log10 estimated CFU/ml for 16S rRNA, tmRNA, pre-16S rRNA, and rpoB, respectively. The RNA loss was independent of inactivation temperature. These findings show that heat inactivation could obviate the need for category 3 laboratories to perform RNA-based testing of TB samples.


Mycobacterium tuberculosis ; RNA preservation; heat inactivation; molecular tests

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