Abstract
Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.
Highlights
Tuberculosis (TB) is a top-ten cause of death worldwide, with an estimated 10 million new cases leading to nearly 1.5 million deaths yearly
Emitted light molecule probes. (ii) Its importance for pathogenesis suggests that it will be expressed during infection. (iii) The human genome does not encode a homologue of Hip1. (iv) Prior work from our group has identified an amino acid recognition sequence that is cleaved by Hip1.33 Here, we describe a new diagnostic probe for detecting Mycobacterium tuberculosis (Mtb): fast, luminescent, and affordable sensor of Hip[1] (FLASH)
Similar results were obtained when comparing the RIF-susceptible strain CDC1551 to the RIFresistant strain (Figure S5C). These results show that FLASH can be used to monitor growth inhibition by clinical antibiotics and can differentiate susceptible from resistant strains of Mtb after 5−6 days of culture with antibiotic substantially faster than the weeks to months generally required for culture-based drug susceptibility testing (DST), and faster than newer methods for DST such as microscopic-observation drug-susceptibility (8 days)[42] and Sensititer MycoTB plates (10−14 days).[43]
Summary
Tuberculosis (TB) is a top-ten cause of death worldwide, with an estimated 10 million new cases leading to nearly 1.5 million deaths yearly. In 2018, there were half a million new cases of rifampicin (RIF)-resistant TB.[1] Early identification of resistance is important for developing proper antibiotic regimens for patients, yet drug susceptibility testing (DST) to determine resistance in clinical isolates can take from 10 days in liquid medium to 28−42 days on solid medium. Molecular tests such as Xpert MTB/RIF provide a rapid method of identifying the presence of mutations that confer resistance, but the design of these tests requires genetic information for each mutation.
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