Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples

Abstract

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2–99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7–100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

“…h) and provides onboard data analysis to enable distributed applications. In other work, we have validated the combined hardware and Zika virus diagnostic platform with 268 patient samples, in parallel with RT-qPCR, and found a diagnostic accuracy of 98.5% 24 . Taken together, our goal is to enable the technology transfer of this platform to researchers so that it can be repurposed and improved by the community to address unmet diagnostic needs.…”

“…We do our best to abate this risk through careful laboratory practice. In a recent clinical trial of 268 patient samples, we did not encounter any contamination issues; however, it is an important consideration 24 . With this in mind, the protocol As we have seen during the COVID-19 pandemic, the instruments used to perform RT-qPCR can themselves serve as a bottleneck and limit patient access to testing.…”

“…1. The portable plate reader device (Figure 8) can be used as an alternative to a conventional plate reader 24 . For the protocol and representative results, see Supplementary Protocol.…”

“…To monitor colorimetric cell-free assays, we enable on-site quantification of results in regions where thermal cyclers are not available. The hand-assembled plate reader called Portable, Low-Cost, User-friendly, Multimodal (PLUM; hereafter referred to as portable plate reader) is also introduced here 24 . Initially developed as a companion device for cell-free synthetic toehold switch diagnostics, the portable plate reader offers an accessible way to incubate and read results in a high-throughput manner, providing integrated, computer vision-based software analysis for users.…”

“…After a 1 h incubation, the reactions were added to cell-free PURExpress reactions on paper discs at a ratio of 1:7.The fold change after 130 min at 37 °C is plotted. This figure has been reproduced from24 . Please click here to view a larger version of this figure.…”