Highly sensitive Ebola test targets 'Achilles' heel' of the deadly disease

April 7, 2021
A new Ebola test could change the testing game. (Christopher Black/World Health Organization via AP)

A new Ebola test could change the testing game. (Christopher Black/World Health Organization via AP)

A new test for Ebola provides a result in 60 minutes and detects infections in monkeys earlier than other assays, according to U.S. researchers who say the technology could help thwart deadly virus outbreaks.

In a study published April 7 in Science Translational Medicine, the team of researchers describes an easy-to-use test that doesn't need to be sent to a lab to produce results. The test has not yet been tested in people but did detect low levels of an Ebola virus protein from a drop of human blood.

Ebola is a severe disease that causes fever, bleeding and organ damage. In 2014-16, an Ebola epidemic in West Africa killed more than 11,000 people, and new cases continue to be reported from ongoing outbreaks in Guinea and the Democratic Republic of Congo. 

Cheap, fast and accurate testing is a mainstay of combating infectious diseases such as Ebola, but currently available tests for the virus don't check all of these boxes. 

"The earlier you can diagnose [patients with Ebola], the earlier you can treat them," said Ashutosh Chilkoti, senior author of the study and a professor of biomedical engineering at Duke University. Ebola is usually treated with antibodies, although antiviral drugs have also shown promise.

"If you diagnose people early, you can triage people and you can contain the disease. So the idea is you want a really sensitive test because the more sensitive [the test], the earlier you can diagnose it," Chilkoti added. 

The gold standard of testing for many diseases, including Ebola, is PCR. This technique amplifies tiny amounts of genetic material, making it highly sensitive at detecting low viral levels. But PCR is expensive, requires trained personnel and special lab equipment and can take days to provide a result. 

Another type of test called lateral flow assays, which work like a home pregnancy test, detect Ebola proteins from a drop of blood, providing a result in just 15 to 30 minutes. Several of these rapid tests for Ebola have been approved by the U.S. Food and Drug Administration, but they often fail to detect the virus at low infection levels, or yield high false-positive rates. A third category of test is rapid cartridge-based PCR tests, which are accurate and fast, but can cost upward of $20 per test. 

To develop a sensitive, low-cost rapid Ebola test, Chilkoti and his colleagues tweaked their previously described D4 assay to detect an Ebola protein called secreted glycoprotein, or sGP. According to Chilkoti, Ebola pumps out lots of sGP early on during infection, which the virus may use as a decoy to fool the immune system. The researchers suspected that sGP could also be Ebola's "Achilles' heel," because the protein could be targeted for diagnostics, Chilkoti said.

The Ebola D4 assay consists of a glass slide with 24 chips. Printed onto each chip are spots of sGP-specific antibodies, which Chilkoti likens to a piece of bread in a sandwich. When a drop of blood is added, sGP proteins bind to these antibodies, forming the "meat" of the sandwich. Elsewhere in the chip are spots of another type of antibody with a fluorescent label encased in a layer of sugar. 

When blood is added, the sugar dissolves and the antibodies are released, allowing them to bind to the other side of the sGP protein, "completing the sandwich," Chilkoti explained. Once the blood is rinsed away, the quantity of sGP protein can be estimated by measuring the fluorescence intensity with a handheld device.

Using human blood spiked with different concentrations of sGP, the researchers found that the test could detect the protein at lower limits of 0.01 nanograms per milliliter in 60 minutes, making it 600 times more sensitive than a lateral flow assay made with the same antibodies. The D4 test also performed equally well with several different virus species that cause Ebola.

To examine the test's performance in detecting Ebola infections in animals, the team inoculated 10 rhesus macaque monkeys with Ebola and drew blood from a subset of the animals each day. The test detected the virus as early as one day after inoculation, while PCR did not detect the virus until three days after inoculation. This suggests the D4 test could be more sensitive than PCR, although Chilkoti acknowledges that the sample size was small.

Another benefit of the test is that it remains stable for months without refrigeration, making it suitable for shipping around the world and point-of-care testing.

"I would love this technology and other technologies to democratize access to health care globally. Because health care in most of the world is pretty decentralized: People don't go to hospitals, they go to the neighborhood doctor or a little clinic," Chilkoti said. "There's no reason that fairly sophisticated tests that were formerly the province of the first world and hospitals and fancy laboratories should not move out of there and go to the masses, go to the people, go where they're needed across the world."  

Chilkoti says his team's assay could easily be adapted to test for other diseases and to screen for multiple diseases at once. The researchers have not yet shown that the test can detect Ebola infections in humans, and they plan to conduct clinical trials in the future. 

"But first, we want to test it in the field, put it through its paces under real-life conditions," he said. Because it's tricky to obtain approval to work with Ebola, the team will conduct field trials with a D4 assay tuned to detect liver disease. 

Each D4 test is estimated to cost around 25 cents, making it cheaper than Ebola lateral flow assays, which cost 50 cents or $1 per sample. But unlike these assays, the D4 test also requires a fluorescence reader. According to the study, commercial readers cost around $150,000, but the researchers describe a new handheld device they dubbed the D4Scope. Composed of off-the-shelf components, the D4Scope prototype costs $1,000 to make.

The study, "Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR," published April 7 in Science Translational Medicine, was authored by Cassio M. Fontes, Jason Liu, Daniela F. Cruz, Garrett Kelly, Kelli M. Luginbuhl, Daniel Y. Joh, Jacob Heggestad, Angus Hucknall, Maiken H. Mikkelsen, Roarke W. Horstmeyer and Ashutosh Chilkoti, Duke University; Barbara D. Lipes, Aiwei Yan, Patricia Shi, Carl F. Pieper and Michael D. Gunn, Duke University Medical Center; and Krystle N. Agans, Stephanie L. Foster and Thomas W. Geisbert, University of Texas Medical Branch at Galveston.

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