Paper-based diagnostic avoids false positives from Dengue fever and other related viruses.

MIT researchers have developed a paper-based test that can diagnose Zika infection within 20 minutes. Unlike existing tests, the new diagnostic does not cross-react with Dengue virus, a close relative of the Zika virus that can produce false positives on many Zika tests.

This test could offer an easy-to-use, cheap, and portable diagnostic in countries where Zika and Dengue are both prevalent and the gold-standard test that measures viral RNA in the bloodstream is not available.

“It’s important to have a single test that can differentiate between the four serotypes of Dengue and Zika, because they co-circulate. They’re spread by the same mosquito,” said Kimberly Hamad-Schifferli, an associate professor of engineering at the University of Massachusetts at Boston, a visiting scientist in MIT’s Department of Mechanical Engineering, and a co-senior author of the paper.

The researchers worked with scientists around the world to test the new device on patient samples and confirmed that it can accurately distinguish Zika virus from related viruses.

Lee Gehrke, the Hermann L.F. von Helmholtz Professor in MIT’s Institute for Medical Engineering and Science (IMES), is also a senior author of the study, which appeared in the September 27 issue of the journal Science Translational Medicine. The paper’s first authors are IMES research scientist Irene Bosch and Department of Mechanical Engineering postdoc Helena de Puig.

One of the biggest challenges in diagnosing Zika is that many of the tests are based on antibodies that interact with a viral protein called NS1, which is found in the bloodstream of infected patients. Unfortunately, many other viruses from the same family, known as flaviviruses, have similar versions of NS1 and can produce a false positive. Flaviviruses include West Nile virus and the virus that causes yellow fever, as well as Dengue virus.

In an effort to create a more precise diagnostic, the MIT team set out to find antibodies that would interact exclusively with NS1 protein produced by the Zika virus, as well as antibodies specific to NS1 from each of the four different strains of the Dengue virus.

To achieve this, the researchers exposed mice to Zika and Dengue viruses and then screened the resulting antibodies, in pairs, against every flavivirus’ version of the NS1 protein. This allowed them to identify pairs of antibodies that react only with one version of NS1 and not any of the others.

“We knew by informatics analysis that if we looked enough, and we teased out the repertoire of the B cells of these animals, we would eventually find those antibodies,” Bosch said. “We were able to tease out the very few antibodies within the repertoire that would give you uniqueness in the detection.”

The researchers used these pairs to create five separate tests, one for each virus. They coated strips of paper with one antibody from each pair, while the second antibody was attached to gold nanoparticles. After adding the patient’s blood sample to a solution of these nanoparticles, the paper strip is dipped into the solution. If the target NS1 protein is present, it attaches to the antibodies on the paper strip as well as the nanoparticle-bound antibodies, and a colored spot appears on the strip within 20 minutes.

This approach requires five test strips per sample to test for each virus, but the researchers are now working on a version that would test for all five with one strip.

Most countries where Zika and Dengue are prevalent do not allow patient samples to be shipped out of the country, so the researchers traveled to several countries, including Mexico, Colombia, India, and Brazil, to test their devices with patient samples.

They found that their results were comparable to those obtained by polymerase chain reaction (PCR) te