A portable, low-cost test for Zika virus may soon be available to doctors

The test successfully detects the virus in monkeys, and researchers are now looking towards product development for distribution to local clinics.

keith_pardee_bwResults published today in Cell Press indicate that a new low-cost, portable test successfully detects Zika virus in monkeys. Currently, doctors working in rural areas hit hard by the Zika outbreak have no way of quickly and accurately testing patients for the virus, as reliable diagnostic methods rely on specialized equipment and trained technicians. The test, a simple “color change” assay, indicates the presence of the virus in blood or saliva. Keith Pardee is one of the researchers on the project, which is headed by Harvard University’s Wyss Institute. We asked him how the test works and how doctors and patients will benefit from it.   

ResearchGate: What are the limitations of currently available Zika testing procedures, and how does the test your group is developing improve on them?

Keith Pardee: A standard method for diagnostics are serological assays, which look at the presence of antibodies for a certain pathogen. However, because the Zika virus is closely related to other viruses that share a very similar geographic range, there’s a lot of cross-reactivity in a serological assay. People who previously had dengue infections, for example, could come up as positive for Zika. Because of this, molecular-based diagnostics that use nucleic acid sequences are more accurate, because they don’t have this problem. But these methods generally require skilled technicians and specialized and costly equipment that’s not portable. Samples have to be collected in the field and brought to labs in urban areas that have these capabilities. And so our aim was to produce a tool that people will hopefully eventually be able to use to do on-the-spot testing in local healthcare clinics rather than only in large, urban centers.

RG: What stage are you at now with the project?

Pardee: This is still very much proof of concept. Through the paper we progressively increase the stringency—trying to get closer and closer to mimicking the way the test would be used in the field—and our final figure shows data from infected monkey plasma. These are monkeys from Dave O’Connor’s group at the University of Wisconsin – Madison, which has infected macaques with Zika virus. They’re doing their own work with them, trying to figure out when the viral loads peak and doing other research, but they also share samples with outside researchers like us. So that’s the closest we’ve got to real samples: plasma samples from monkeys.

RG: How does the test work?

Pardee: With the work flow we used, you take a small volume of the plasma and heat it to 95 degrees for two minutes. This breaks the virus open and releases the RNA. We then add the sample to the first of the three molecular tools that make up the system. This first step is an amplification step, which can take a really low concentration of RNA in a patient sample and bring it up to the level that the second tool, the RNA sensor, can detect. The technical name for that is a toehold switch, a programmable RNA sensor that’s a riboregulator. We can program these things to detect basically any sequence, and they produce a color change that is visible to the eye when positive. In this paper we developed 48 of them, targeting 24 regions in the Zika virus genome, and then picked the top performers. Ultimately we hope these will be used in the field along with a third molecular tool based on CRISPR, or Cas9, that can be used to distinguish the strain of the virus at a single base pair resolution. If a patient did test positive with the first RNA-based sensor for the Zika virus, this third tool can distinguish between the African and the American strains of the virus.

All of those tools can be freeze-dried: Cas9, the amplification system, and the transcription/translation system that runs the RNA sensor. That means they can be distributed without refrigeration. The amplification is a liquid step, and that liquid is used to rehydrate the paper disc that has the RNA sensor. Similarly, the Cas9 genotyping tool is a liquid-based step in which the product is used to rehydrate those paper discs. Ultimately, we want to combine all these tools in one freeze-dried paper disc, but given the urgency of the Zika outbreak, that isn’t the top priority right now.

RG: How did you ensure the accuracy of the test?

Pardee: We exposed our molecular tools to RNA sequences from the dengue virus at low and high concentrations, as well as off-target regions of the Zika genome. The sensors can not only distinguish between viruses, but also wouldn’t be turned on by variants we weren’t looking at in the genome of the Zika virus. Specificity is of course something that would be even more closely examined by product development experts.

RG: Now that you have proof of concept from testing in monkeys, what’s the next step?

Pardee: Now that we’ve done the proof of concept, we need to find the resources—either in the public or private sector—to fund product development, so the test can be made available to practitioners on the front lines of this global health crisis. That would involve making the test more user friendly, things like embedding the instructions in a way that someone in a clinical lab could run it. The goal is to get to the product out of the realm of academia and into the hands of professional diagnostics researchers for development and manufacturing.

RG: Can you estimate a timeline for that?

Pardee: Ideally on a scale of months. It’s hard to say for sure at this point, but we’re definitely seeing interest in the technology.

RG: Why is developing a test that can be administered in the field important for addressing the Zika outbreak? 

Pardee: Like everyone, we were moved by the images of children with microcephaly and also hearing about the other serious neurological problems that are associated with this American strain of the virus. We feel it’s important to get tools out that may help reduce the virus’s impact until a vaccine can be developed. On a meta level, this kind of test could help national and global health organizations track the outbreak. It will of course also make it easier for healthcare providers to identify the people who are affected, treat them as fast as possible, and take precautions so they aren’t a source of further spread.

RG: How did you become involved in the project?

Pardee: This was a multi-institutional effort from seven different institutions. It was led by Jim Collins of the Wyss Institute at Harvard, and many of us are either former or current post-docs and students of his.

Featured image: A Wyss Institute researcher holds a cartridge containing the paper-based diagnostic. The discs that appear purple indicate a sample infected with Zika virus. Yellow indicates a negative result. Courtesy of the Wyss Institute at Harvard University.