An international research team led by Harvard Medical School and MIT scientists has 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.

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

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.

The findings were published in the September 27 issue of the journal Science Translational Medicine.

“When we have traveled to the places where these viruses are problems, the people there unanimously say that they need more surveillance. They need to know which viruses are circulating in their environments,” said senior study author Lee Gehrke, professor of health sciences and technology in the Department of Microbiology and Immunobiology at HMS, and the Hermann von Helmholtz Professor in the Institute for Medical Engineering and Science at MIT.

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, the virus that causes yellow fever and dengue virus.

In an effort to create a more precise diagnostic, the 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.

“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 Boston and co-senior author of the paper.

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.

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 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 to 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 in patient samples.

They found that their results were comparable to those obtained by polymerase chain reaction (PCR) tests, which detect viral RNA in the bloodstream. PCR tests are not widely used in areas where the virus is found because they require trained personnel and lab equipment that are not available everywhere. Dengue infects hundreds of millions of people annually, mostly in