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Proteomic Test to Diagnose, Differentiate Zika, Dengue Developed by A*STAR Team


NEW YORK (360Dx) – Researchers from  Singapore's Agency for Science Technology and Research (A*STAR) have reported a new mass spectrometry-based diagnostic assay that they said can detect and differentiate multiple flaviviruses, including Zika and dengue.

The targeted proteomic test works by tracking a particular secreted flaviviral protein, known as nonstructural protein 1 (NS1) — which is unique for each virus and serotype — allowing the assay to detect multiple flaviviruses, as well as viral co-infections. 

In a study published this week in Proceedings of the National Academy of Sciences, the researchers reported 100 percent specificity in distinguishing different flaviviruses, which also include yellow fever and Kunjin viruses. The test can also differentiate between dengue serotypes; Zika and yellow fever are associated with only one virus serotype.

What's more, senior author Jayantha Gunaratne of A*STAR Institute of Molecular and Cell Biology told 360Dx that the assay "offers many advantages over other diagnostics, including the ability to diagnose and determine multiple flavivirus types over a longer diagnostic window." Current diagnostic methods, which include antibody- and PCR-based tests, cannot offer all these capabilities in a single assay, he said.

The research was done in collaboration with the Singapore Environmental Health Institute and National Centre for Infectious Diseases.

While the researchers are not immediately pursuing commercialization, the assay could be clinically useful: PCR-based tests require viral RNA, which decreases in abundance four to five days after the onset of clinical symptoms. The proteomic test, however, can detect flaviviruses over the first eight days following the onset of symptoms.

"Hopefully, this prototype method may be transformed into a more rapid point-of-care test," Pei-Yong Shi, a flavivirus researcher at the University of Texas Medical Branch who was not involved with the research, told 360Dx. "You may be able to immediately tell if they're infected with dengue." He added that mass spec technology could be the basis for a cost-effective clinical test, but noted that the test's sample preparation method and instrumentation need to be further improved.  

The need for flavivirus tests

In recent years, Zika and dengue have become global concerns as they've spread. According to the World Health Organization, 84 countries show evidence of mosquito-borne Zika virus. Meanwhile, there were approximately 3 million suspected or confirmed cases of dengue in 2015.

The prevailing methods used to diagnose and monitor flaviviruses rely on antibody and PCR technologies. "PCR-based assays are used to directly detect existing viruses in patient blood," Shi said, while "antibody-based serology assays are used to diagnose infections after the viruses have already been cleared from the patients."

Tests for dengue and Zika targeting the NS1 protein also already exist, but the antibodies often cross react with proteins from the other flaviviruses, making it difficult to differentiate between them. Moreover, many existing dengue tests do not distinguish between different serotypes, although in 2018 a team from Columbia University reported a peptide microarray-based assay capable of serotyping dengue. 

The NS1 protein is highly conserved and in some cases, peptide sequences between virus serotypes or closely related viruses differ by only a single amino acid. Additionally, the authors noted that NS1 is an attractive target because "it is secreted as early as the first day of illness and stably present in the patient blood" for up to nine days.

The A*STAR assay is different from other tests targeting the NS1 because it detects the protein by its amino acid sequence, rather than antibodies designed to bind to the protein.

Using bottom-up proteomics, the researchers began by obtaining NS1 peptides specific to dengue serotypes. The assay was able to serotype 70 percent of 96 patient serum samples with 100 percent specificity, while a PCR-based assay was only able to serotype 50 percent of them.

The assay uses parallel reaction monitoring, a high-resolution mass spec technology. "We employed a nano-LC system coupled to a mass spec instrument with a quadrupole for targeted peptide isolation and a high mass accuracy and resolution detector," Gunaratne said. The researchers analyzed trypsin-digested peptides from sera and secretomes using either a ThermoFisher Q-Exactive or Q-Exactive Plus mass spectrometer. "These features contributed to the significance of our results," Gunaratne noted.

Once they had demonstrated the ability to detect dengue and differentiate between serotypes, the team developed the assay to include other flaviviruses, using similar methods to select peptides. The team tested the multiplex assay on Huh-7 cell suspensions infected with Zika, yellow fever, Kunjin, and dengue viruses.

The researchers said they also used the full flavivirus parallel reaction monitoring assay (fvPRM) on 12 Dengue-infected patient serum samples and "observed precise diagnosis and serotyping with no cross-reactivity." 

The authors also noted that they have the ability to easily extend the assay for new flaviviruses or serotypes and could accommodate at least 50 targets. They also said their test could differentiate between virus genotypes (for example, Zika has two genotypes, African and Asian/American) although they did not validate this using patient samples. 

Drawbacks of the test include "the need for skilled personnel and costly [mass spectrometry] instrumentation," the authors said.

Shi noted that any assay that directly detects the flavivirus or its proteins has limitations, because the infections are acute. "By the time someone has symptoms, the virus is already decreasing," he said, and by the time a patient gets tested, the infection may be hardly detectable. "In clinical labs, particularly in public health diagnostic labs, they're very interested in detecting the [human] antibodies the virus generates," he said. "Obviously, this assay doesn't address that challenge," he said.

Gunaratne said the team has not yet determined the costs of running and developing the assay, nor has it formed a company to commercialize the test or made plans for clinical trials.