A low-cost, easy-to-use, rapid diagnostic test for diagnosing Zika and dengue viruses and differentiate between the four serotypes of dengue virus has been developed. It has high sensitivity and specificity for both dengue and Zika viruses.
An international team, including researchers from India, has developed a low-cost, easy-to-use, rapid diagnostic test for diagnosing Zika and dengue viruses and differentiate between the four serotypes of dengue virus. None of the rapid tests currently available is capable of differentiating the four dengue virus serotypes.
If the kit is used for diagnosing dengue cases, it would not cross react with any of the other flaviviruses (Zika, Japanese encephalitis virus and West Nile virus).
Materials costs for the assay worked out to nearly $5 per strip, and the scientists speculate that the price will decrease as production is scaled up. India alone accounts for nearly 35% of the estimated dengue cases in the world.
Since Zika virus and dengue virus are related flaviviruses, many proteins are similar in both these viruses. While many diagnostic tests cannot strictly distinguish between Zika and dengue infections, the test developed by the international team has nearly 100% ability to distinguish the two virus infections. The results were published in the journal Science Translational Medicine.
High sensitivity and specificity
The diagnostic test has nearly 76-100% sensitivity and specificity in the case of dengue, while the sensitivity is 81% and specificity is 86% in the case of Zika. “The sensitivity and specificity values may improve once the kits are validated with larger number of samples,” says Dr. Guruprasad Medigeshi from the Translational Health Science and Technology Institute (THSTI) in Faridabad and one of the authors of the paper. “Since the antibodies recognise the antigens which are specific to dengue and Zika viruses there will be no cross-reactivity leading to wrong diagnosis.” Dr. Medigeshi is an Intermediate Fellow of the Wellcome Trust DBT India Alliance.
Dengue and Zika transmitted by Aedes mosquito co-circulate in many countries, India included. Hence, it is important to be able to differentiate between the two virus infections. Also, the ability to quickly identify the dengue serotype is important for disease management from public health point of view.
Infection by one serotype supposedly produces lifelong immunity only against that specific serotype; it does not protect against other serotypes. Infection for the second time by a different dengue serotype leads to increased infectivity and more severe clinical manifestations such as dengue haemorrhagic fever and dengue-shock syndrome. This kind of aggravation of the infection when infected for the second time by a different dengue virus serotype is called antibody-dependent enhancement (ADE).
How the test was developed
The researchers injected specific flavivirus nonstructural 1 (NS1) proteins produced by Zika and dengue viruses into mice to generate monoclonal antibodies. Despite the close similarity between different flavivirus NS1 proteins, the researchers identified pairs of antibodies that can specifically detect and distinguish each of the four dengue serotype NS1 proteins as well as the Zika NS1 protein.
They used the antibody pairs and coated each antibody on a strip of chromatography paper at two different spots. One of these antibodies is attached to gold nanoparticles. “When a serum sample from a patient is added on the chromatography paper where the antibody is spotted, the antigen present in the serum binds to the first antibody. This way the virus antigen is first captured. Since it is paper, the antigen bound to the antibody diffuses and comes in contact with the second antibody. The second antibody too binds to the antigen leading to the formation of colloidal aggregates of gold nanoparticles, which then forms a pink spot,” says Dr. Medigeshi. A pink spot appears on the strip within 20-30 minutes after the second antibody binds to the captured antigen.
What the pink spot indicates
The appearance of the pink spot indicates positivity to either Zika virus or dengue virus. And in the case of a serotype test, it indicates the respective dengue virus serotype.
“Since each pair recognises a particular serotype we need four strips for dengue serotyping and one strip for Zika for testing each sample,” says Dr. Medigeshi who had helped in the validation of the kits and analysis of the data.
The monoclonal antibodies developed by these groups recognise 15-20 amino acids on NS1 proteins that are either unique to each dengue serotype or similar in all dengue serotypes but different from Zika virus.
The team has developed a pan-dengue strip which indicates positivity to dengue serotypes without cross-reacting with Zika NS1 unlike the current diagnostic kits tested in the study. The pan-dengue strip will help in knowing if the sample is positive to dengue virus but cannot differentiate between the four serotypes.