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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.

Author information

1
School of Medicine, University of St Andrews, St Andrews, United Kingdom ws31@st-andrews.ac.uk.
2
Instituto Nacional de Saúde (INS), Ministério da Saúde, Maputo, Mozambique.
3
Division of Infectious Diseases and Tropical Medicine, University of Munich, Munich, Germany.
4
German Centre for Infection Research (DZIF), Partner Site Munich, Munich, Germany.
5
School of Medicine, University of St Andrews, St Andrews, United Kingdom.

Abstract

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.

KEYWORDS:

Mycobacterium tuberculosis ; RNA preservation; heat inactivation; molecular tests

PMID:
30728191
PMCID:
PMC6440770
[Available on 2019-09-28]
DOI:
10.1128/JCM.01778-18

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