While it’s no longer dominating the headlines, the Zika virus continues to be a threat around the world. Now, researchers at the New York University College of Dentistry are collaborating with Rheonix to develop a test that uses saliva to identify diagnostic markers of the virus in a fraction of the time of current commercial tests.
“The recent Zika virus outbreak confirms that we need an effective surveillance and diagnostic program to reduce the impact of future emerging infectious diseases,” said Maite Sabalza, PhD, a postdoctoral associate at the Department of Basic Science and Craniofacial Biology at NYU Dentistry.
Early identification is critical for stopping the spread of infectious diseases. Testing often involves two separate steps: one to detect a pathogen’s nucleic acids (RNA or DNA) and another to test for antibodies, the proteins the body produces in response to pathogens.
Blood samples, which most often are used to test for Zika virus, are typically processed using a real-time polymerase chain reaction (RT-PCR). But the virus disappears in the blood a week or two after infection, while it can persist longer in saliva, semen, and urine. Also, antibodies can remain for months or years in those bodily fluids.
The new test combines both nucleic acid and antibody assays using saliva, since the Zika virus and antibodies both persist in the fluid. Saliva tests also are noninvasive, cost-effective, and easier to collect than blood or urine. Plus, they have the potential to produce results in a matter of minutes instead of days.
Current RT-PCR tests take around three hours, and specific antibody tests can take several weeks. The researchers are using a different method called isothermal amplification, which can detect a virus’ nucleic acids in as little as 20 minutes, and antibody tests that can take less than an hour using Zika-specific antigens.
“The sooner you can identify a pathogen, the sooner steps can be taken to treat and isolate people. During an epidemic, you could test people before they get on a plane. The future of going through security at the airport may not be taking off your shoes, but instead spitting into a tube,” said Daniel Malamud, PhD, professor of basic science at NYU Dentistry.
The NYU researchers are building on their earlier collaborative work with Rheonix in developing a rapid saliva test for HIV that can detect both viral RNA and antibodies.
“When we developed the HIV test, we knew we could use the same model for any infectious disease. All we need to know is the nucleic acid sequence and an antigen to identify specific antibodies,” said Malamud.
The researchers then altered this model to use markers that detect Zika nucleic acid sequences instead of those for HIV. They then used a portable isothermal amplification device, which could be used for point-of-care testing, to identify Zika RNA.
After showing that the test could confirm the presence of the virus using both purified Zika RNA and Zika-infected saliva, the researchers adapted the test to the Rheonix CARD cartridge and workstation, enabling them to estimate the viral load in saliva samples. Knowing the amount of virus present helps clinicians understand disease severity and immune responses, particularly among pregnant women and fetuses.
The researchers also identified Zika-specific antigens, which can be used to detect the Zika-specific antibodies needed for testing, using a high-density peptide microarray. Because Zika can be confused easily with other viruses such as Dengue and Chikungunya, identifying Zika-specific antigens paves the way for more precise diagnostic testing.
With evidence showing that both the nucleic acid and antibody tests work, the researchers can combine them in the Rheonix CARD cartridge to process both diagnostic assays automatically and simultaneously.
“In working towards the goal of quickly making effective diagnostics available, we have developed a generic protocol that can not only be used to test for Zika virus but can be adapted for the next emerging or re-emerging infectious disease,” Sabalza said.
“In addition to being able to react very quickly to any newly emerging or re-emerging infectious disease, our fully automated testing platform requires virtually no hands-on efforts and is well suited for use by individuals with minimal training,” said Richard Montagna, PhD, senior vice president for scientific and clinical affairs at Rheonix.
The research was presented in “Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva” in PLOS One and in “Zika Virus Specific Diagnostic Epitope Discovery” in the Journal of Visualized Experiments.
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