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Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory

Palekyte, Ana; Morkowska, Anna; Billington, Owen; Morris-Jones, Stephen; Millard, James; Marakalala, Mohlopheni J; Owolabi, Olumuyiwa; ... Honeyborne, Isobella; + view all (2024) Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory. Clinical Chemistry 10.1093/clinchem/hvae013. (In press). Green open access

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Abstract

BACKGROUND: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. METHODS: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. RESULTS: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). CONCLUSIONS: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

Type: Article
Title: Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1093/clinchem/hvae013
Publisher version: https://doi.org/10.1093/clinchem/hvae013
Language: English
Additional information: Copyright © Association for Diagnostics & Laboratory Medicine 2024. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Infection and Immunity
URI: https://discovery.ucl.ac.uk/id/eprint/10189472
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