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-Guerin (BCG) cultures and TB-positive sputum samples, we show that boiling for 20 min at 80, 85, and 95 degrees 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-165S rRNA, and rpo8 by reverse transcriptase quantitative PCR (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA species dependent, <1 log(10), 1.1 log(10), 1.3 log(10), and 2.4 log(10) estimated CFU/ml for 16S rRNA, tmRNA, pre-16S rRNA, and rpo8, 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.
Item Type: | Journal article |
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Faculties: | Medicine |
Subjects: | 600 Technology > 610 Medicine and health |
ISSN: | 0095-1137 |
Language: | English |
Item ID: | 81326 |
Date Deposited: | 15. Dec 2021, 14:58 |
Last Modified: | 15. Dec 2021, 14:58 |