NEW YORK (360Dx) – A team led by researchers at the Massachusetts Institute of Technology has developed a dipstick test for diagnosing Zika virus from patient serum.
The test, which is also able to distinguish between different dengue virus serotypes, could enable rapid, inexpensive testing for Zika and dengue infections, said Lee Gehrke, professor of health sciences and technology at MIT and one of the developers of the test.
He added that he and his colleagues have applied for patents covering the test and are in discussions with regulatory agencies as well as commercial entities interested in bringing it to market.
In a study published on Wednesday in Science Translational Medicine, the test detected Zika with sensitivity of .81 and specificity of .86 while detecting the four dengue serotypes with sensitivity and specificity ranging from .76 to 1.00.
Zika has emerged in recent years as a major public health crisis, with infections associated with birth defects like microcephaly and the development of Guillain-Barré syndrome. Dengue, meanwhile, has long been a challenge in infectious disease, with, according to the STM study authors, hundreds of millions of infections occurring annually and more than a fourth of the world's population at risk for the disease.
Both Zika and dengue are flaviviruses and can be detected by measuring the presence of the flavivirus nonstructural 1 (NS1) protein in the blood of infected patients. Commercial tests using NS1 to diagnose dengue currently exist, but, the authors noted, the antibodies used in these tests have been demonstrated to cross-react with Zika NS1 proteins, making it difficult to distinguish between the two diseases.
Additionally, existing rapid tests do not distinguish between different dengue serotypes, which is significant because one of the four dengue serotypes does not necessarily confer immunity to infection with the other three.
While methods like PCR are able to diagnose diseases like Zika and dengue with high specificity, the resource-constrained environments where Zika is a major challenge often don't have access to the technology, said MIT researcher Kimberly Hamad-Schifferli, coauthor on the STM study and also a developer of the test.
"We thought of this as something that could be distributed or deployed widely in endemic areas, because a lot of the places that have dengue and Zika often don't have access to clinics, and so they don't have access to PCR machines," she said. "It often will take a week to get lab results back, and that's simply too long. We wanted to make something that could be used on the spot, with no additional reagents, and that a non-expert could use on themselves, essentially, for a diagnosis."
The test uses a basic immunochromatography format essentially like that of a home pregnancy test. Key to making it work was generating and identifying antibody pairs specific to the different forms of the NS1 protein. To do this, the researchers generated candidate antibodies by injecting groups of mice separately with recombinant NS1 from the four dengue serotypes and Zika, and then used ELISA to screen these antibodies against the different NS1 proteins, as well as proteins linked to other diseases including West Nile, Yellow Fever, and Japanese Encephalitis.
The relative binding observed in these ELISAs established the different binding of each antibody to each NS1 protein type, and using this information the researchers selected 11 dengue antibodies and 10 Zika antibodies for further evaluation. They then tested these antibodies against cells infected with Zika or different dengue serotype, using flow cytometry to assess their binding to their intended targets, as well as their cross-reactivity to NS1 proteins not intended to be targeted.
They then incorporated these antibodies into a dipstick format, testing them in different combinations (726 for dengue and 300 for Zika) to arrive at the eight dengue antibodies and two Zika antibodies they ultimately used in their assays.
Gehrke said that while screening candidate antibodies against a range of potentially cross-reactive proteins associated with other diseases might seem an obvious approach, it is not commonly done.
"It was a really large amount of work to do the screening the way we did it," he said, noting that he and his colleagues originally planned to use commercially available antibodies in their test but that "all of the antibodies that we looked at that were commercially available strongly cross-reacted and were not at all useful for us."
He added that screening against markers for multiple infectious diseases allowed for identification of antibodies that could prove useful in diagnosing those other diseases.
Hamad-Schifferli added that, in fact, the researchers began the work before the recent Zika outbreak and were initially focused on developing a test for distinguishing between dengue serotypes.
"We were able to react nimbly and generate a test for Zika, as well," she said. "And what we've learned can be applicable to other outbreaks that occur in the future."
The team tested the diagnostic in a retrospective study of samples from human subjects collected in Brazil, Mexico, Colombia, Panama, and Guatemala that had been validated as positive or negative for dengue or Zika using reverse transcription PCR with positives further validated via ELISA. The Zika test correctly identified 25 of 31 positive cases for 81 percent sensitivity and six of seven negative cases for 86 percent specificity.
These results are on the order of other clinical tests for infectious disease, Gehrke said, adding that he and his colleagues are now working with regulatory agencies to define the structure of additional trials.
The clinical samples needed for this work "are difficult to obtain," he said, "so we are getting all of our ducks in a row, so we know what is required for the regulatory approvals."
The researchers are also in discussions with commercial entities about bringing the test to market, as well as with governmental agencies in countries including Brazil and India that are charged with bringing low-cost medical care to their populations.
"We hope that within a year or so we're positioned to start bringing these tests to the people who really need them," Gehrke said.