Detecting Zika in Mosquitoes? Technology Being Developed As Alternative to Human Blood Testing

NEW YORK (360Dx) – A team of researchers in Australia, Brazil, and the US is field testing a technique that detects arboviruses, including Zika, in mosquitos to help pinpoint virus hotspots before the infected insects ever contact humans.

The technique — which is based on near-infrared spectroscopy, or NIRS — is 18 times faster and 110 times cheaper than the use of quantitative reverse transcription polymerase chain reaction, a technique with potential for pathogen screening in mosquitoes, but one that's too expensive for the large-scale analysis of mosquitoes needed for surveillance programs, Maggy Sikulu-Lord, a coauthor of the study and researcher at the University of Queensland, in St. Lucia, Australia, said in an interview.

Clinicians have been using diagnostic testing of patient blood to help mitigate the effects and spread of the Zika virus, but the use of NIRS can find the virus in mosquitoes before it spreads to humans, Sikulu-Lord said.

The researchers published the results of an investigation earlier this month in the journal Scientific Advances that describes the use of NIRS as a rapid, reagent-free, and cost-effective tool that can be used to detect the Zika virus in heads and thoraces of intact Aedes aegypti mosquitoes. "We have demonstrated that we can detect the Zika virus in mosquitos with prediction accuracies of 94 to 99 percent," Sikulu-Lord said.

The technology has potential to make a difference in disease surveillance, potentially stopping outbreaks and saving lives, the researchers said. Further, according to Sikulu-Lord, the researchers are developing the technique to distinguish among several similar infections including dengue and chikungunya in addition to Zika. The researchers also anticipate expanding on NIRS to identify potential arbovirus hotspots, and to guide the spatial prioritization of vector control.

They are doing this work in response to the accelerating global spread of arboviruses, such as Zika, which highlights a need for more proactive mosquito surveillance, the researchers said. Rapid and affordable tests that can screen for large numbers of infected mosquitos in a specific region are currently unavailable, and that presents a major challenge to preventing mosquito-borne diseases, they added.

Sikulu-Lord and her team began using NIRS to detect the age and species of mosquitos, a parameter useful in identifying those that could be infected and in guiding treatments. They then decided to develop it as an alternative to molecular techniques to detect the Zika virus in the Aedes aegypti, or yellow fever, mosquito, a common vector of arboviruses.  

Researchers have used NIRS for decades for chemical analysis in agriculture, pharmaceuticals, and medicine. The method classifies biological samples based on the type and the concentration of chemical compounds in samples. Using the method in a Zika diagnostic instrument, the researchers shine a beam of light directly on a mosquito and then collect and analyze its diagnostic spectrum components.

Alternate techniques that rely on RT-qPCR or ELISA testing are time-consuming and expensive when you need to analyze large numbers of mosquitoes, according to Sikulu-Lord. They also are impractical in the field, where resources and equipment can be limited.

Other available rapid diagnostic tests for arbovirus detection in mosquitoes rely on the capture of viral antigens through an enzyme-linked immunosorbent assay that uses reagents, which in turn require refrigeration that's often unavailable in low-resource settings. Investigators have proposed the use of honey-baited nucleic acid preservation cards to capture arboviruses from mosquito saliva and to eliminate the problem of testing individual mosquitoes, but the technique still requires RT-qPCR to test for viral RNA, the researchers said.

In another effort aimed at finding an alternative method, researchers at the University of California, Riverside, are developing an electronic nanobiosensor that uses single-walled carbon nanotube networks as part of a chemiresistor transducer functionalized with anti-dengue NSI monoclonal antibodies for rapid detection of dengue nonstructural protein 1, or NS 1. They reported successfully detecting NS1 in spiked adult Aedes aegypti homogenate over a broad dynamic range with high sensitivity and selectivity.

Partly because of challenges with existing tests, arbovirus surveillance programs in endemic countries often rely solely on human case reports, a strategy that has proven inadequate for containing arbovirus outbreaks, Sikulu-Lord and her colleagues noted. Routine pathogen surveillance in mosquitoes to identify regions that are hotspots of transmission would require testing thousands of mosquitoes each month, which has not been implemented because available techniques are too costly, Sikulu-Lord said.

Although a near-infrared spectrometer capable of doing this kind of testing can cost about $60,000, one could use it to test at least 60,000 mosquitos for infection in a year, and its operating costs are near zero, she said, adding that conducting the same number of tests using RT-qPCR would cost about $600,000.

For their study, Sikulu-Lord and her co-researchers collected adult female Aedes aegypti mosquitos and transported them to a lab to test for the virus. The researchers fed half of the population with blood infected with Zika virus and the other half with uninfected blood. The NIRS instrument collected spectra from the mosquitoes' heads, thoraces, and abdomens, and effectively distinguished these infected body parts from uninfected mosquitoes seven days after infection.

According to Sikulu-Lord, a near-infrared spectroscope lends itself to being easily transported from one location to another, and her team has moved their method into field testing.  

Given the technique’s rapid, high-throughput, and reagent-free nature, hundreds of samples can be processed in a day by unskilled technicians, enabling rapid predictions of potential disease transmission, which in turn could facilitate a rapid action plan to stop major disease outbreaks, the researchers said.

Their study follows a report published this month in the New England Journal of Medicine suggesting that current screening for Zika virus in donated blood using PCR-based methods may be a poor use of resources.  

Collaborators from the American Red Cross, Grifols Diagnostic Solutions, Quality Analytics, and the Wadsworth Center at the New York State Department of Health evaluated testing results of more than 4 million individual US blood donations during a 15-month period in 2016 and 2017 and concluded that screening for Zika virus nucleic acids was "costly and had a low yield."

Similar to their own study, this one points to the need for more effective methods of detection than are currently being applied, Sikulu-Lord said.

She also pointed to an earlier study that she said underscores the importance of identifying multiple arbovirus sources in specific regions. In it, a group of international researchers analyzed symptomatic case reports from the city of Merida, Mexico, with the goal of assessing the utility of historical dengue virus data to infer chikungunya and Zika virus introduction and propagation. Writing in the journal PLOS Neglected Tropical Diseases in March, the group said that their study opens "a window for considering historical [dengue virus] data to make predictions of likely sources of invasion for other emerging Aedes-borne viruses, as well as to the consideration of spatially-targeted approaches for delivery of vector control and surveillance."