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Colorado State LAMP Method Promises Easier, Less Expensive Zika Testing


NEW YORK (GenomeWeb) – A team led by investigators at Colorado State University has devised a new assay to directly detect Zika virus from mosquitoes and from a variety of unprocessed clinical sample types like blood, saliva, and semen.

The approach uses a simple nucleic amplification method called LAMP (loop-mediated isothermal amplification) that could make it possible to deploy tests to areas without the resources for the complex and expensive technologies necessary for current PCR-based molecular tests.

In their study, published today in Science Translational Medicine, the investigators reported that their assay can not only detect Zika virus in mosquitoes and human bodily fluids with comparable sensitivity to quantitative PCR methods, but also that it could distinguish between African and Asian strains, a potential aid in efforts to more effectively track the virus’ spread.

The LAMP method also avoided any false positives for closely-related pathogens such as dengue and Chikungunya viruses, the authors reported.

Joel Rovnak, the study's senior author and an assistant professor in Colorado State University's Department of Microbiology, Immunology and Pathology, said this week that he and his team will now using funding from the National Institute for Allergy and Infectious Diseases to perform a head-to-head comparison study with collaborators at a pediatric hospital in Managua, Nicaragua, putting the LAMP assay up against a standard PCR method.

That project is slated to begin later this year. If successful, it would provide important validation data to support translation of the assay to a clinical test for humans.

Commercialization of a test for human use either in the US or abroad isn't something that Rovnak and his team are focused on, however.

"The basic technology for LAMP is patented and licensed, so what we brought really is a new application and the primers we designed, but we're not patenting those either," he explained. "A pharma company or one of the companies that already does RT-PCR, they could adopt this and license it from the patent holders for LAMP, but that's not something we are doing." Japan's Eiken Chemical owns the rights to the LAMP method.

The CSU researchers are instead planning to devote their energy to advancing the approach in mosquito surveillance and disease control.

Specifically, they plan to test their LAMP assay against PCR methods in mosquito and wildlife samples from Puerto Rico obtained through a project with the US Department of Agriculture's Animal and Plant Health Inspection Service.

Rovnak said that the goal that prompted the team's study in the first place was to develop an assay for testing mosquito samples that really lowered cost and complexity as much as possible.

"The majority of the countries involved in the current outbreak are not rich, [so] it's important for us to try to develop low-cost surveillance methods," Nunya Chotiwan, a Colorado State University grad student and the study's first author, said in a statement.

For example, the CSU group estimated that a heating block for LAMP costs at most $250 — compared to real-time PCR machines that cost between $15,000 and $25,000.

"My personal dream is that there would be an unspecialized person ... deployed to spray pesticide for instance [deployed to] a lakeside or a reservoir or a community [who could] confirm that they do have in front of them these positive mosquitoes," Rovnak said.

This contrasts with how eradication efforts are currently guided, Rovnak said. "Last year for example they did aerial bombardment of Puerto Rican cities with Naled, which is a pesticide that is not approved in the US or Europe."

"If the economics were right you could potentially deploy a lot more surveillance and you could be much more targeted," he added.

Rovnak and some co-authors on the current study were turned on to the potential of a LAMP-based Zika test after developing a LAMP assay for pneumonia in wild sheep.

"We were funded by Colorado parks and wilderness to develop what we call a 'sheep-side assay,' so that investigators in the field could [tranquilize] dart sheep from trucks or helicopters, take samples from their nostrils and run the LAMP from the truck in a heat block," Rovnak explained.

In the case of Zika, it seemed that the technology could support a test cheap and easy enough to be deployable in a similar style. "If we can get this into the back of a pickup truck with a mosquito sprayer, that's the idea," he said.

Although tests for mosquito-borne viruses have previously been developed using a version of LAMP that involves purifying RNA and adding reverse transcriptase to the reaction, Rovnak and his colleague's approach uses the reverse transcriptase activity of Bst DNA polymerase itself for direct detection of viral RNA in mosquito or human samples without the need for RNA isolation.

In addition, the team showed that they could perform the test on direct samples, without first extracting or isolating RNA.

According to the authors, the resulting test detected virus down to the lowest levels that have been observed in a Zika virus-infected insect, which represents enough sensitivity to detect a single infected mosquito in a collection pool of 50 non-infected mosquitoes.

Although human testing wasn’t the initial goal of the CSU researchers, they did show strong initial results in the study that suggest their LAMP method could compete strongly against other tests that are now used in humans.

Using Zika virus artificially spiked into samples from healthy individuals, as well as in clinical specimens collected from confirmed cases of Zika virus infection, the team showed that the assay allowed detection at sensitivities high enough for acute infection settings.

"However, assays of clinical samples present challenges to the direct detection of Zika virus RNA by the LAMP assay," the group wrote, since the requirement for 100-fold sample dilution limits the sensitivity of the direct LAMP assay.

However, this dilution factor is not necessary for LAMP amplification from purified RNA. And indeed, "when matched RNA preparations were used, the LAMP assay without reverse transcription matched RT-PCR with respect to specificity and levels of detection," the authors wrote.

Though Rovnak and his colleagues aren't actively pursuing clinical development of a test for humans using LAMP, there is definitely interest among other parties. Researchers from the University of Pennsylvania, for example, recently published their own reverse-transcriptase LAMP strategy for Zika testing using microfluidic chips.

Commercial firm Rheonix also recently received funding to develop an RT-LAMP and immunoassay system for Zika testing.

A variety of groups, like Sandia National Labs and AI Biosciences, have also been developing low-cost, field-deployable molecular testing systems that could be applicable to Zika.

Nanobiosym Diagnostics also obtained Emergency Use Authorization last month for a Zika assay that is now permitted for laboratory use on the firm's 3.5-lb. handheld Gene-Radar test platform.

However, validation of that test was performed against traditional tests in a lab setting, so the company does not currently have authorization to use it outside of a lab, the firm said at the time.