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Glowing dye simplifies TB detection
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Researchers have constructed a molecule that can illuminate Mycobacterium tuberculosis in sputum samples within 1 hour. Preliminary study findings published in Science Translational Medicine suggest a diagnostic method that uses the molecule to detect tuberculosis may be simpler than current microscopy-based methods.
According to Carolyn R. Bertozzi, PhD, professor in the department of chemistry at Stanford University and investigator at Howard Hughes Medical Institute, and colleagues, rapid TB diagnostics currently used in high-burden countries include the color-based Ziehl-Neelsen test and the fluorescent auramine-based Truant stain. Both tests involve staining the mycomembrane that surrounds M. tuberculosis to identify infected cells. The protocols require “extensive processing” to remove dye from surrounding bacteria while retaining dye in mycobacteria, the researchers noted. The sensitivity of these tests ranges from 32% to 94%, depending on the method and experience of the user. Therefore, newer tools are “urgently needed” to streamline the testing process and improve diagnostic accuracy, they said.
Previous research has shown that sugar molecules known as trehaloses are unique to mycobacteria and corynebacteria, but not gram-positive or gram-negative organisms. Therefore, Bertozzi and colleagues hypothesized that a staining method targeting trehaloses may be useful in specifically flagging M. tuberculosis but not other bacterial species. They combined trehaloses with a light-emitting chemical known as DMN to see whether the hybrid molecule — which the researchers termed “DMN-Tre” — would illuminate M. tuberculosis. A labeling method using DMN-Tre to identify M. tuberculosis cells was tested on 16 sputum samples obtained from treatment-naive patients with TB. The sensitivity and efficiency of DMN-Tre labeling was compared with a standard testing protocol using Auramine O stain.
According to the researchers, both tests performed similarly in terms of the number of M. tuberculosis cells detected. However, DMN-Tre labeling required only a single incubation step without washes, making it “considerably simpler than the multistep auramine procedure.” Results from DMN-Tre labeling were available in less than 1 hour.
In additional experiments, DMN-Tre did not illuminate gram-negative or gram-positive pathogens, confirming that the method is specific for TB. Further investigations revealed that the intensity of fluorescent labeling decreased within hours of exposure to TB treatment, which means DMN-Tre labeling could be used for drug sensitivity testing.
“Although our results suggest that DMN-Tre can distinguish metabolically active vs. inactive organisms, further studies are warranted to assess whether DMN-Tre distinguishes live vs. dead cells,” the researchers wrote. “It will be particularly interesting to determine whether nonreplicating, or ‘dormant,’ [M. tuberculosis] cells are detectable with this method.”
Another benefit of DMN-Tre is that it can be stored at room temperature for weeks, according to the researchers.
“Thus, the DMN-Tre labeling procedure may translate well both to research and to clinical applications in low-resource environments,” they concluded. – by Stephanie Viguers
Disclosures: Bertozzi and two other authors are inventors on a U.S. patent application submitted by Stanford University for solvatochromic dye conjugates.
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