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Stanford Group Publishes Field Evaluation of Zika, Chikungunya, Dengue Triplex Assay

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NEW YORK (GenomeWeb) – Researchers at Stanford University Medical School have published a triplex assay to detect Zika, chikungunya, and dengue viruses in a single patient sample. The group has also begun discussions with various commercial diagnostics manufacturers with the hopes of collaborating to bring its ZCD test through US Food and Drug Administration Emergency Use Authorization (EUA).

As described in a July online article published ahead of print in Emerging Infectious Diseases, the researchers evaluated the Stanford ZCD test in various sites in Nicaragua, and used it to detect the first case of Zika virus infection in that country.

The test is now being used for epidemiological purposes in Nicaragua to screen national surveillance samples for febrile illnesses, Benjamin Pinsky, an infectious diseases researcher at Stanford University Medical School and corresponding author on the study, told GenomeWeb in an interview. A collaborator in Ecuador is also using the test for clinical purposes, Pinsky said.

He also noted that the Stanford group is now in discussions with a number of companies to try and bring the ZCD test through the FDA EUA process, but he declined to provide additional details at this early stage.

Zika symptoms overlap significantly with those of dengue and chikungunya. All three are transmitted by mosquitoes. While dengue can be most deadly, chikungunya can cause long term physical debilitation and Zika has been associated with neurological syndromes and microcephaly in babies born to mothers infected in the first trimester of pregnancy.

Although there are no drugs to treat these viral infections, untangling which one, or ones, a patient has can guide clinicians in determining how to monitor patients. It is also important for epidemiological tracking. The Zika outbreak in Central and South America and the Caribbean has led to more than 1,000 cases of microcephaly, according to the World Health Organization, and is expected to reach the US mainland this summer.

The US Centers for Disease Control and Prevention has also created a test for these targets called Trioplex, which has received EUA.

Pinsky said his group out of necessity compared the Zika target in the ZCD test to a previously published CDC test, referred to as the Lanciotti assay, since the agency has not yet disclosed the primer-probe sets for its Trioplex assay. 

However, "based on the analytical performance characteristics presented in the Emergency Use Authorization documentation [for the Trioplex assay], the test that we're performing is approximately 10-fold more analytically sensitive," Pinsky said.

The lower limit of detection of the Trioplex assay, at least for Zika, is right around the mean value for virema, Pinsky said. So, in his estimation, the CDC test is "likely to miss quite a number of cases since the level of viremia for Zika is statistically significantly lower than for dengue and chikungunya."

The ZCD test demonstrated a linear range of 108 to 10 copies for Zika virus and DENV-3, according to the EID study. For DENV-1, -2, and -4 and for chikungunya, the range was 108 to 100 copies. In copies per milliliter, the 95 percent lower limit of detection for each target was 7.8 for Zika virus; 13.2 for chikungunya; 11.7 for DENV-1; 13.5 for DENV-2; 4.1 for DENV-3; and 10.5 for DENV-4.

On 216 patient samples tested in Nicaragua, the ZCD assay found 56 Zika virus-positive samples.

The assay was also shown to be non-reactive to other pathogens, such as West Nile, Japanese encephalitis, tickborne encephalitis, yellow fever, Saint Louis encephalitis, o’nyong-nyong, Semliki Forest, Mayaro, Ross River, Getah, Barmah Fores, and Unas viruses.

The Stanford team has been developing the Zika portion of its test since 2014, as part of a larger program to develop infectious diseases assays, Pinsky said.

"As part of these assays, we were looking to make multiplexes that contained potentially emerging infectious diseases, particularly arboviruses," he said. The group had been using these to screen samples from throughout the world, including Africa, Asia, and Central and South America. The Pacific Islands were also a source of specimens, and Zika appears to have changed in some way from its pre-epidemic nature as it passed through that region in 2007. 

The group also previously validated a test for dengue fever, leptospirosis, and malaria, called DLM. That test uses a pan-Plasmodium primer-probe set that detects, but does not distinguish, all five Plasmodium species that infect humans, with a separate P. falciparum-specific primer-probe set that utilizes a different fluorescent label, allowing a call-out for P. falciparum. The dengue assay for the DLM test is the same as is in the ZCD test, Pinsky said.

The ZCD assay has so far been tested on a variety of platforms, including Thermo Fisher Scientific's ABI 7500, Qiagen's Rotor-Gene Q, Bio-Rad's CFX96 and IQ5, and Roche's LC480.

The group currently has a number of ongoing studies on Zika epidemiology and viral load in a variety of patient groups, and it is also now looking at the virus in mosquitoes.

The CDC recently updated its guidance to suggest that urine be used to test patients with suspected Zika infection, and Pinsky said the ZCD test can accommodate that. "We have a study ongoing in Nicaragua to determine the performance characteristics on that specimen type," he said. 

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