Field-based patient trial for cell-free Zika testing delivers highly accurate results

An international team of researchers, headed by experts from the University of Toronto’s Leslie Dan Faculty of Pharmacy, has led one of the first field trials for a synthetic biology-based diagnostic using patient samples. This work, conducted on-site in Latin America, reveals the potential for cell-free synthetic biology tools and companion hardware for providing rapid, de-centralized, and low-cost patient testing for infectious diseases like the Zika virus.

Study results, published in Nature Biomedical Engineering, show that the novel diagnostic platform has analytical specificity and sensitivity equivalent to a US Centre for Disease Control PCR test for Zika and a diagnostic accuracy of 98.5 per cent with 268 patient samples collected in Recife, Brazil. The platform is also programmable and can be similarly applied to detect any pathogen sequence. In addition to validating highly accurate diagnostic results for Zika, the team also achieved similar diagnostic performance for chikungunya virus, another mosquito-borne arbovirus.

“We see emerging diagnostics, like the paper-based tests we’ve developed, as having tremendous near-term potential to augment existing PCR capacity, improve equity in access to health care, and aid in the responses to public health crises,” said Keith Pardee, associate professor in the department of pharmaceutical sciences, Leslie Dan Faculty of Pharmacy, University of Toronto.

Portable diagnostics aim to improve future outbreak response

Prior to the current global COVID-19 pandemic, the 2015/2016 outbreak of Zika virus in Latin America emphasized the urgent need for rapid and low-cost testing that can be deployed beyond the reach of centralized diagnostic labs, explains Pardee who has a Canada Research Chair in Synthetic Biology and Human Health. “We were investigating and developing this technology well before the COVID-19 pandemic brought these issues to light at the global level. We’ve now been able to apply it and validate it in a region of endemic disease, which is really promising because these tools are meant to enable health systems to better respond to future outbreaks of infectious disease, particularly in low-resource settings,” he said.

Researchers from the international team led by experts from U of T's Leslie Dan Faculty of Pharmacy test the portable diagnostic technology onsite in Recife, Brazil

Dr. Lindomar Pena, department of virology, Oswaldo Cruz Foundation (Fiocruz), led the Brazilian team that collaborated on the project. "This robust diagnostic platform displayed desirable features to be used in developing countries such as Brazil and in laboratories with basic infrastructure. We hope it can be further developed and deployed in the Brazilian network of public health laboratories to diagnose Zika patients, trace contacts and identify hot-spot areas with active community transmission,” he said.

The portable diagnostic platform is a combination of a cell-free, paper-based test and a field-ready companion device that allows data to be collected using image-based color analysis – purple for positive and yellow for negative.  Called “PLUM” (Portable, Low-cost, User-friendly, Multimode), the toaster-size reader presents results from up to 384 samples and displays them in a single image capture. The hardware and software that make up PLUM were originally developed by co-authors Livia Guo and Seray Çiçek as part of their graduate work in the Pardee lab. To keep production costs low, Guo and Çiçek, co-founders of LSK Technologies, used customizable software programs and off-the shelf electronics, enabling PLUM to be built for approximately $500 USD per unit.      

On the molecular side, the cell-free tests can be freeze-dried, allowing for distribution without refrigeration and, significantly, all of the molecular components of the test are independent of the PCR-supply chain. “Here we have demonstrated that these two technologies combined create a low-cost, highly accurate diagnostic tool,” said study lead author Margot Karlikow, a postdoctoral fellow in the Pardee lab from 2016 to 2021 and now co-founder of En Carta Diagnostics.  “We also demonstrated that it is feasible to transport the platform across a significant distance and implement it effectively in another country. In many low- and middle-income countries, there is no PCR testing available outside of main cities, so the ultimate goal is that this platform be used as a high-quality alternative to PCR in more regional settings,” she said. As part of their publication, the team has shared a roadmap of how they were able to transport the diagnostic technology from Canada to Latin America so that other teams might benefit from their experiences.

U of T Entrepreneurship community supports health start-ups

Guo, Çiçek and Karlikow successfully moved their work with the Pardee lab to establish health tech startups, LSK Technologies and En Carta Diagnostics, respectively. The entrepreneurship and incubator community at U of T was instrumental in this process.

In particular Guo and Çiçek, collaborated closely with H2i and UTEST. “Both H2i and UTEST were great in terms of making introductions and helping to expand our network,” said Çiçek, explaining that H2i helped the team figure out the road map from the regulatory and product design standpoint and UTEST helped with customer discovery so they could better understand and mitigate customer pain points.

“The entrepreneurship community at U of T helps foster regular and valuable discussions between entrepreneurs around their challenges,” said Karlikow who is now running En Carta Diagnostics from Paris, France. “With so much support available, it helps you to make the decision to start your company to push your vision and product forward,” she said.

Tackling the next challenges

Showing that the platform could be transported and accurately detect Zika virus in patient samples is a significant step forward in creating more accessible and de-centralized testing, says Pardee. However, the extraction of RNA from patient samples still requires liquid handling by skilled technicians at this stage. “With performance on patient samples now validated, we are tackling these next challenges, like sample preparation, so that the platform and PCR-like diagnostic capacity can be distributed more broadly into the communities where they are needed.”

“This was more than just the science involved, we made it applicable. We took it to the real-world, and it worked.”

The momentum from achieving this successful field-based patient trial will continue to move the team forward. “This step of translating the technology from the lab and applying it in a real-world setting was so important,” said Karlikow. “This was more than just the science involved, we made it applicable. We took it to the real-world, and it worked.”