Simple Test Could Improve TB Diagnosis in Developing Countries

Researchers have developed an innovative diagnostic assay for tuberculosis (TB) that may replace the complicated error-prone test often used in resource-limited areas. Field trials of the experimental new test began in June 2016 in South Africa, which has a high incidence of TB.

The researchers, led by Prof. Carolyn R. Bertozzi, PhD, Stanford University (Stanford, CA, USA), presented their work at the American Chemical Society’s (ACS; Washington, DC, USA) 252nd National Meeting & Exposition, held August 21-25 in Philadelphia, PA, USA.

In wealthier countries, a patient suspected of having TB can be examined with a chest X-ray or a patient sputum or saliva sample can be tested by modern techniques such PCR. But in developing nations with limited resources and spotty access to electricity, samples are often checked for TB with the Ziehl-Neelsen (ZN) test, developed in the 1880s. The procedure takes several hours and is even not very sensitive, missing some TB cases and resulting in many false positives.

Years ago the researchers began investigating bacterial cell wall glycolipids involved in causing TB. Each glycolipid consists of the sugar trehalose linked to a lipid. They discovered that if they provided slightly modified forms of trehalose to the bacteria, the microbes would metabolize and integrate them into their glycolipids. Other researchers showed that the bacteria can take up forms of trehalose attached to a fluorescent dye molecule. A cell that picks up these sugars glows green. “We thought we could use this to detect the bacteria in sputum samples,” said Prof. Bertozzi. Unfortunately, the other researchers’ dye also sticks to other components in saliva, making it difficult to distinguish the bacteria.

The team solved this problem by attaching trehalose to a “solvatochromic” dye that doesn’t glow until it’s incorporated into the cell walls. As a result, there is no background glow. In addition, the procedure is relatively simple: the technician takes a sputum sample, squirts a small amount of dye mixture onto it, and after an hour examines it under a microscope. Even better, whereas the ZN test dyes label both live and dead cells, the new test labels only live cells as it depends on the bacterial cells metabolizing & integrating the trehalose. Since the ZN test cannot determine whether the number of live cells is decreasing, it cannot be used to monitor treatment effectiveness. “If the drugs aren’t working, you want to switch the patient to the next treatment as quickly as possible so you don’t contribute to drug resistance,” said Prof. Bertozzi.

Prof. Bertozzi’s team is also studying other fluorescent dyes that may work even better, and using their current trehalose/dye molecule to explore the structure and properties of the TB bacteria’s cell wall.

Related Links:
Stanford University
American Chemical Society