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

Sabiiti, Wilber; Azam, Khalide; Esmeraldo, Ezembro; Bhatt, Nilesh; Rachow, Andrea und Gillespie, Stephen H. (2019): Heat Inactivation Renders Sputum Safe and Preserves Mycobacterium tuberculosis RNA for Downstream Molecular Tests. In: Journal of Clinical Microbiology, Bd. 57, Nr. 4, UNSP e01778-18

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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 0095-1137
• Sprache: Englisch