Simple test improves diagnosis of TB in developing nations

The researchers will present their work at the 252nd National Meeting & Exposition of the American Chemical Society (ACS).

Worldwide, almost 10 million people become ill with TB, and nearly 2 million die from the disease each year, according to the World Health Organisation. The worst-hit regions include Southeast Asia and Africa.

In wealthier countries, a patient suspected of having TB can be examined with a chest X-ray. Or a sample of the patient's sputum, or saliva, can be sent to a lab for testing by techniques such as polymerase chain reaction (PCR).

But in developing nations with limited resources and spotty access to electricity, patients are often checked for TB with the Ziehl-Neelsen (ZN) test, which was developed in the 1880s.

Technicians using this 11-step procedure put a saliva sample on a microscope slide, then dye it and rinse it multiple times. The process takes several hours. Even worse, "the ZN test is not very sensitive. It misses some cases of TB, and it gives a lot of false positives," says Carolyn R. Bertozzi, Ph.D. These limitations led Bertozzi and her team at Stanford University to develop a new test.

But that wasn't the researchers' initial goal when they began studying TB 16 years ago. At the time, they were investigating molecules, known as glycolipids, in the cell walls of the bacteria that cause the disease. Each glycolipid consists of the sugar trehalose linked to a lipid, or fat.

The researchers discovered that if they provided slightly modified forms of trehalose to the bacteria, the microbes would metabolise the sugar molecules and integrate them into their glycolipids.

Other researchers showed that the bacteria can take up forms of trehalose in which each sugar is 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," Bertozzi says.

Unfortunately, the other researchers' dye also sticks to other components in saliva, making it tough to distinguish the bacteria.

Bertozzi's 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's no background glow. In addition, the process couldn't be easier: The technician takes a sputum sample, squirts a little of the dye mixture onto it, and then after an hour looks at it under a microscope to see if anything is glowing.

Even better, only live bacterial cells can metabolise the trehalose/dye molecules. The dyes in the ZN test, however, label both live and dead cells. That means the traditional test can't determine whether the number of live cells is decreasing, so it can't be used to tell whether a patient's treatment is working."

"Because many strains of TB bacteria are now resistant to the standard treatments, "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," Bertozzi explains.

The group is now working with a collaborator in South Africa to see how the new test performs in real-world conditions. In the meantime, Bertozzi's team is studying other fluorescent dyes that could work even better in a TB test.

They are also using their current trehalose/dye molecule to explore the molecular structure and physical properties of the cell wall of TB bacteria. That knowledge could shed light on the bacteria's drug-resistance mechanism, as well as potential new ways to kill the cells.