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Is it Zika or Dengue? New Test Seeks to Clear up Confusion

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NEW YORK (360Dx) – During the most recent Zika epidemic, just getting the right diagnosis proved challenging: Clinicians had trouble telling whether a patient might have been infected with Zika or something similar from the same family of viruses.

But researchers from the University of Texas Medical Branch and the New York State Department of Health have developed a test that they say may change that by offering better specificity over existing tests and eliminating any confusion about a diagnosis. Soon, the NYS health department will begin using the microsphere immunofluorescence assay (MIA) at its Wadsworth Center Laboratory to test for Zika in patients, it said.

During the Zika epidemic that swept through the Americas starting in 2015, clinicians trying to diagnose patients who may have been infected with the virus were sometimes left scratching their heads. While tests could determine whether a patient had been exposed to a flavivirus, they could not discern whether the person had Zika or another infection from the same genus.

Flaviviruses is a group of viruses that include along with Zika, dengue fever, yellow fever, West Nile virus, and others that can manifest similar symptoms in patients. Importantly, immunoassays, one of the two most common Zika testing technologies, also cannot differentiate one flavivirus from another due to a lack of specificity.

This is where the new test, described in a recent study in EBioMedicine, may prove clinically useful, its developers said.

Based on current guidelines from the US Centers for Disease Control and Prevention, Zika tests use one of two technologies — RT-PCR, which detects the virus directly from blood; and immunoassays, which detect antibodies produced by the body after it is exposed to the virus.

While RT-PCR has high specificity and sensitivity, it has a narrow timeframe during which it can be used, Pei-Young Shi, a professor of biochemistry and molecular biology at UTMB and one of the authors of the EbioMedicine study, said in an interview.

In blood, the Zika virus is typically detectable for only up to 10 days after a patient is symptomatic, he said, after which RT-PCR tests will no longer be able to detect the virus, Shi said.

Indeed, the World Health Organization recommends that RT-PCR testing be done on serum samples that have been collected no longer than three days after symptoms first appear. With saliva samples, testing should be done within a five-day window.   

Further compounding the problem is that many people infected with the virus are asymptomatic, and if they show symptoms, by the time they manifest, the viral load has already dropped "significantly. So, usually when a patient presents himself or herself to the physician …  the virus will be gone," Shi said.

After that window closes, serological assays — which work by picking up immunoglobulin M antibodies to the Zika virus — are used, instead, to diagnose possible Zika infection. Because the antibodies stay in the body for much longer than the virus, the technology can offer a diagnosis even months after a patient was initially infected.  

"The strength of this [type of] assay is it's very sensitive," Shi said, but, such assays have low specificity, and further, the body's immune response to flavivirus structural proteins are "very cross-reactive."

As a result, serology tests can tell whether someone has been infected with a flavivirus, but not necessarily which flavivirus.  

"So, it cannot give the physician a definitive answer," Shi said.

The US Food and Drug Administration also recently flagged another problem with serological IgM tests after Laboratory Corporation of America reported false-positive results with the first commercially available Zika serological IgM test, manufactured by Inbios International, to diagnose patients.

In response, the FDA recommended that physicians not rely solely on the results from that test to make a diagnosis.

According to Shi, though, low specificity is the primary problem of serological tests in flavivirus testing.

To address this, he and his colleagues began by asking themselves a series of questions about other proteins that may be made by the virus during infection, including whether there could be antibodies "that are made or are elicited by those other proteins," and whether those antibody reactions could be virus type-specific, he said.

He explained that structural proteins form the virus particles, but another type of protein called non-structural proteins are not components of the virus particle. Instead, they are made during the viral infection of the cell. Shi and his co-researchers hypothesized that these non-structural proteins could be used to differentiate one flavivrus from another.

To test this, they developed an assay with seven proteins — the structural Zika envelope protein, widely used in other Zika assays; the non-structural proteins Zika NS1 and Zika NS5; and recombinant dengue NS1 proteins from each of the four serotypes. 

For the testing platform, they chose Luminex's technology, rather than an ELISA format, so that they could perform multiplexing.

They tested their assay on samples from 153 patients known to be infected either with Zika, or dengue, or both. If the patient sample reacted to one of the Zika non-structural proteins, it meant that the patient was infected with Zika, and if the patient sample reacted with the dengue non-structural protein, it meant the patient was infected with dengue. If the patient sample reacted with both the Zika and dengue non-structural proteins, then it suggested the patient had been exposed to both viruses.

"Using this, clearly our data says that [our] assay is better than CDC's current assay," Shi said, adding that the test would have use on all populations, including pregnant women, newborns, and men.  

Susan Wong, director of the Wadsworth Center's Diagnostic Immunology Laboratory, said in an email that the assay has been validated with 300 patient samples, while another 800 samples are also part of the validation study, though not all the results have been completed.

Current CDC guidelines call for positive results from an IgM Zika test to be confirmed via a plaque neutralization assay, which can take more than one week to provide test results. The assay developed by Shi and his collaborators, which provides results in 48 hours, has the potential to sidestep that need because of its higher specificity, Shi said.

"Basically, you can replace all the recommended CDC serology assays in a single new assay," he said.

Wong added that the assay results have been compared against the plaque reduction neutralization testing method, and she and her co-researchers will continue to do so once the new assay is used routinely.

Ranu Dhillon, a faculty member at the division of global health equity at Brigham and Women's Hospital and Harvard Medical School, and who has written about Zika, said in an interview that the most valuable use for Shi's assay might be as a screening tool to identify "Zika hot spots" where exposure to the virus may be especially high, such as Miami, the epicenter of last summer's Zika epidemic in the US. People identified by the assay as being infected would then be recommended for DNA- and RNA-based testing for confirmation.

He added that the test could also be helpful in preventing or alleviating backlogs of test results, such as what happened in Florida during the summer when there were weeks-long delays in laboratories getting test results.

Dhillon further pointed out that there is a need for a point-of-care Zika test in settings without easy access to a laboratory. The new assay does not yet have POC capabilities, but Shi said that it could be developed into a "dipstick-type" POC test, and he and his colleagues are working to move it "toward that direction."

The researchers anticipate submitting the assay to the US Food and Drug Administration for emergency use authorization, followed by possible commercialization efforts, Wong said. Shi added that talks are underway with industry players to potentially commercialize the assay, though he declined to elaborate.

While the proof-of-concept work looked at Zika and dengue, the MIA technology can easily be expanded to include other antigens for differentiating other flaviviruses, which Shi and his collaborators plan to do. That would be further down the road, though, while they work to improve the current assay's ability to detect and differentiate Zika from dengue.

In the US between Jan. 1, 2015 and Feb. 1, 2017, nearly 5,000 Zika cases were reported in the 50 states, with another 36,000-plus Zika cases reported in the US territories, according to the CDC.

While the Zika epidemic has largely subsided, it has not disappeared. Texas late last month, for example, confirmed a non-travel-related case of a pregnant woman getting infected with the virus. As long as Zika remains a threat, the need to develop a better diagnostic tool for it remains, Shi said.

"The current available landscape has major flaws and that needs to be improved. So, in that sense, it's never too late," he said. "We still don't know next year what the situation will look like."