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Zika Dx Development Process Could Provide Roadmap for Other Tests, Rheonix, Collaborators Say


NEW YORK (GenomeWeb) – Researchers at Rheonix and the New York University College of Dentistry have developed a nucleic acid and antibody-based test for Zika virus.

Described in a pair of recent studies, one in PLOS One, the other in the Journal of Visualized Experiments (JoVE), the test enables rapid saliva-based testing for the virus while also providing information on the timing of infection that could prove useful for clinicians, particularly in the treatment of pregnant women, said Daniel Malamud, a professor of the NYU College of Dentistry and author on the papers.

Perhaps more notable is the process underlying development of the test, which Malamud said can be easily and quickly applied to other emerging infectious diseases in the future.

In fact, the Zika work stemmed from a previously collaboration between Malamud's lab and Rheonix in which the parties developed an HIV test that used Rheonix's CARD cartridge and Encompass Optimum microfluidic system to simultaneously test for antibodies and nucleic acids indicative of the disease.

In HIV, antibodies against the virus typically appear a week to 10 days after infection, while HIV nucleic acid appears earlier. Serological tests are often used as an initial diagnostic with patients, who, if they test negative, are then referred to nucleic acid testing. However, Malamud said, a percentage of these patients never complete this follow-up testing, making a rapid simultaneous antibody-nucleic acid test desirable.

Additionally, in resource-constrained areas, PCR-based testing can be too expensive for widespread use. The NYU-Rheonix assay uses reverse-transcription loop-mediated isothermal amplification (RT-LAMP), which is typically cheaper than real-time PCR.

The Zika assay likewise uses RT-LAMP to detect viral RNA. For the immunoassay portion of the test the researchers used high-density peptide microarrays containing the full ZIKV protein divided into 3,423 unique peptides each consisting of 15 unique amino acids plus a 14-amino acid overlap to search for binders to host Immunoglobulin M (IgM) and Immunoglobulin G (IgG).

Detecting both Zika nucleic acids and host antibodies allows for detection of the disease across its various stages. As in HIV, the nucleic acids appear first with the antibody response arising later, with IgG antibodies appearing first followed by IgM antibodies.

"If you have all three of those things—the nucleic acids, the IgG antibody, and the IgM antibody— you have a timeline for the infection, which is important particularly for pregnant women. You can ask, when were you actually infected, and how do we treat you?" Malamud said.

The speed of the assay is also an improvement over existing tests, he said, particularly with regard to the serological assay. Getting results back from conventional serological assays for Zika can take weeks, he said, compared to less than an hour for the combined nucleic acid-antibody results from the NYU test.

The researchers did much of the initial assay development using samples spiked with Zika but have since begun validating the test in actual patient samples. Thus far, the test has proven to be specific for Zika when tested in patients with infectious diseases like dengue fever that can present similarly. Malamud and his coauthor Maite Sabalza, a postdoctoral researcher at NYU, are now working to validate the test in additional patient samples from Brazil and other countries.

From a commercial perspective, however, the test may turn out to be more of a proof-of-concept than an actual clinical product, said Richard Montagna, senior vice president for scientific and clinical affairs at Rheonix.

Particularly "from the standpoint of US public health, I think most people are of the perception that the [Zika] problem has passed," he said. "Whether it will come back in again as we move into the warmer seasons, we don't know. So it's a real question if, from a market standpoint, is there a market that currently remains?"

That said, Montagna said the company has received inquiries from a number of parties interested in the test.

"We probably would not take it through the FDA on our own," he said. "But we believe that we have the framework in place that, if people who are already in that marketplace are interested, we'd certainly like to talk to them."

More significantly, development of the Zika test, and of the HIV test before it, demonstrated that Rheonix and its NYU collaborators could rapidly develop combined nucleic acid and serological assays for use on the Encompass Optimum platform, Montagna said.

"From our standpoint, I think the most important piece of information is that this process that we've run through was really pretty short," he said. "It was like six to eight weeks, and we were done. So let's assume the next emerging infectious disease comes, or even a reemerging infectious disease comes along. We could react pretty quickly."

The Zika work was an outgrowth of a NIH grant funding development of the HIV assay, Montagna said. The company received that grant, a $1.5 million Small Business Innovation Research Phase II grant, in 2015. In 2016 it received a $656,414 SBIR Phase I/II grant for the Zika work.

Rheonix is currently working with the US Food and Drug Administration on studies to support a submission for its clinical version of the Encompass platform, the Encompass MDx, along with an initial assay— triplex sexually-transmitted infection test for gonorrhea, chlamydia, and vaginalis.

The FDA recently approved a protocol covering internal and external studies of the instrument and test, Montagna said, adding that the company expects to finish its internal studies at the end of April, after which it will begin external trials.

"We've got 12 collection sites set up around the United States, and we have four testing sites where we'll be shipping instruments to have them run the external trials," he said. "So that will be the first test hopefully that will not only give [FDA] clearance to the instrument, but also the assay."