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

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.

inadequate. Studies following the 2016 outbreak found that inadequate surveillance and diagnostic systems contributed to spread of the Zika virus, and that social and economic disparity in access to health services exacerbated the challenges faced by vulnerable populations 14 . Taken together, the shortfall in diagnostic capacity led to calls for molecular diagnostics that can be used at the point of care (POC) and motivated the development of several new Zika diagnostic technologies, many of which came from the field of synthetic biology [15][16][17] .
We were one of the groups that contributed to the effort for new and portable Zika virus diagnostics 15 . Using computationally designed toehold switch-based sensors (Supplementary Note 1) targeting the Zika RNA genome, we developed a paper-based test using cell-free protein expression reactions (PURExpress) that could be freeze-dried for distribution without refrigeration. Containing the recombinant enzymes of transcription and translation from Escherichia coli, these reactions first transcribe the RNA-based toehold switch from a DNA template and then, if the target Zika viral sequence is present, translate a reporter protein (for example, β-galactosidase (LacZ)) to create an optical signal through enzymatic cleavage of chlorophenol red-β-D-galactopyranoside (CPRG, yellow → purple; Fig. 1a) 18 . To reach clinically relevant sensitivity, an isothermal RNA amplification reaction (nucleic acid sequence-based amplification (NASBA)) was placed upstream in the workflow to provide detection down to the low-femtomolar range (3 fM) 19,20 . Notably, all of the molecular components for the test are independent of the PCR supply chain and, if needed, can be fabricated using the infrastructure of a simple microbiology lab 15,18,[21][22][23][24] . These features make this in vitro and biosafe technology well suited for use where there is a need for de-centralized capacity, such as in low-resource settings or during public health crises.
Until recently, portable diagnostic technologies based on synthetic biology have remained largely untested under field conditions with patient samples. To address this need for validation, we assembled a team of laboratories from five countries to perform a patient trial of our paper-based Zika diagnostic devices onsite in Latin America (Fig. 1b) 15 . Here we report field trials for a synthetic biology-based diagnostic using patient samples, and we benchmarked the test performance directly to a US Centers for Disease Control (CDC) RT-qPCR test for the virus 25 . After optimization of the molecular components, and as part of establishing a PCR-free molecular diagnostic at field sites away from the benches of home laboratories in Canada and the US, we developed a low-cost, computer vision-enabled, automated plate reader (which we named the PLUM reader, for portable, low-cost, user-friendly, multimode reader) to perform low-volume, high-capacity optical measurements in a 384-well format. Using RNA extracted from the serum of patient samples, we find that the combined PLUM reader and paper-based Zika sensor provide analytical sensitivity and specificity for the Zika virus equivalent to RT-qPCR, with a diagnostic accuracy of 98.5%. We further show that the combination of molecular, hardware and software tools can be used as an adaptive system for the point of need. In a second proof-of-concept effort, we demonstrate that by simply changing the molecular components that confer specificity to the assay (in particular, NASBA primers and toehold switch), we can detect the chikungunya virus (98.5% accuracy), which is another arbovirus with worldwide distribution that causes significant morbidity [26][27][28] .

Results
Preparation of the molecular tools. We began by optimizing the original paper-based Zika virus diagnostic workflow for distribution to field sites (Extended Data Fig. 1) 15 . This started with evaluation of analytical sensitivity using in vitro-transcribed RNA inputs. As previous work has shown, the incorporation of an isothermal amplification step can be used to provide detection of target nucleic acids well within clinically relevant concentrations 15,17,29 . Using this two-step process, we demonstrated detection of target RNA from  1 | Schematic of the paper-based diagnostic system. a, System development. Virus-specific toehold-switch-based sensors were computationally designed based on the target virus genomic sequence. DNA encoding the toehold switch was then embedded into paper discs with a cell-free system (CFS) that transcribes the RNA-based sensor. In the presence of target viral RNA, the toehold switch enables cell-free translation of the LacZ (CFS: LacZ) reporter gene to create a colorimetric output (yellow → purple). The system is programmable and can be similarly applied to detect any pathogen sequence, enabling the formation of a molecular tools library. b, Field trial. Using cultured virus, the analytical sensitivity and specificity of the paper-based diagnostic were first validated using a two-step NASBA and toehold switch-based method. All of the data were collected and analysed using the in-house PLUM reader. RT-qPCR was performed in parallel for all experiments for comparison. Using the same method, validation was followed by a patient trial using RNA extracted from patient serum samples.
concentrations as low as 1.24 molecules per µl (equivalent to ~2 aM) (Fig. 2a,b and Supplementary Method 1). This is an increase in analytical sensitivity of three orders of magnitude over our previously published results, which was achieved by optimizing primer concentration (500 nM to 12.5 uM) 15 , and, ultimately, we found that reducing the primer concentration (to 500 nM) provided the best sensitivity. Before transferring the assay to the teams in Latin America, we wanted to confirm that the workflow would accommodate the detection of virus-encapsulated target sequences. For this, we chose to test the diagnostic with an engineered lentivirus containing a small segment of the Zika viral genome. Lentivirus was chosen over the Zika virus for safety and practical reasons. Although both viruses are categorized as Biosafety Level 2 (BSL-2), the lentivirus contains only a small portion of the Zika viral genome, reducing risk of disease, and has been engineered with safety features that mitigate many of the risks of working with Zika virus, including replication incompetence 30 . These factors, combined with the lentivirus being a widely used and easy-to-implement research tool, made the virus a useful proxy to evaluate assay performance from the context of encapsulated RNA.
We began by extracting RNA from the lentiviral particles using conventional column purification (Fig. 2a,c) and then tested the ability of the diagnostic assay to detect the lentiviral RNA without the extraction step, using a simple boiling step to lyse the viral capsid for direct assay input (Fig. 2a,d). In both cases, the Zika assay was able to detect the lentivirus down to 10 3 plaque-forming units (PFU) per ml. With the diagnostic assay ready for field testing, the primer sequences, toehold switches and protocols were transferred to laboratories in Brazil, Ecuador and Colombia to establish testing capacity onsite in Latin America.
A portable, high-capacity plate reader. The transition of a paper-based diagnostic assay to field-deployable applications requires a field-ready companion device, and so, with no commercial options for low-cost and portable optical quantification, we developed a portable device that provides quantitative and high-throughput measurements of the paper-based Zika diagnostics onsite. Previous efforts developed companion electronic readers; however, the devices had limited sample capacity (n < 16) and required a laptop and separate incubator for operation 15,22 . Moreover, these designs required a dedicated light-emitting diode (LED)/sensor pair (US$6.50) for each sample, which alone would make the deployment of the high-capacity system needed for a patient trial impractical and costly for a 384-well format.   Fig. 2). The red line represents a theoretical diagnostic threshold that was determined using sensitivity data from field trials.
illumination source for the PLUM reader (Supplementary Note 2). To achieve an even illumination over the plate, a custom-built, 3D-printed light box housing the LED board was assembled by using a layer of tracing paper as a diffuser. To confirm that the design was free of positional measurement effects, triplicate cell-free expression reactions with and without LacZ reporter were run at five locations in a 384-well plate (Fig. 2f,g). The level of variation across the 15 parallel positive-reaction and 15 negative-reaction time-course measurements taken by the PLUM reader was modest (Fig. 2g) and compared well to measurements taken of a replicate plate using a commercial plate reader ( Supplementary Fig. 2, bottom).
Optical measurements. Optical absorbance measurements are a routine and key functionality for plate readers and are central to measurement of the LacZ reporter output for the Zika virus diagnostic (570 nm). To measure absorbance, conventional plate readers directly monitor the intensity of the wavelength of interest and create an absorbance value by inverting the quantified decrease in transmitted light signal ( Supplementary Fig. 3a) 32 . However, without the sophisticated components of conventional plate readers, such as monochromators and optical slits, we needed an alternative approach. Our solution was to use computer-vision-based software to compensate for the simplicity of the PLUM reader's components (for example, camera-based measurement; Supplementary Fig. 3b).
Here, rather than measuring absorbance directly, we use all the light reaching the RGB channels of the camera to measure the spectral colour shift caused by optical changes in the sample ( Supplementary Fig. 2, left column). Using the colour additive theory, the resulting algorithm calculates absorbance-equivalent values by taking a ratio of the increasing colour channel over the decreasing colour channel value 33,34 . During each experiment, an image of the plate is collected at each capture interval (for example, 5 minute periods), and the RGB channel values for all well regions of interest are stored in a data file (for example, CSV). To quantify the purple reporter signal of Zika-positive samples, onboard software calculates the ratio of blue channel values over green channel values, providing similar absorbance-equivalent outputs ( Supplementary  Fig. 2). Once done, using well-assignment data from the plate map, data can then be automatically analysed, graphed (Fig. 2g), uploaded via the Internet to the cloud storage (Amazon Web Services S3) and shared worldwide with collaborators.
Field trial of the diagnostic test for the Zika virus. After molecular and hardware training onsite in Latin America, we evaluated the performance of the Zika virus diagnostic system using cultured viruses (analytical specificity and sensitivity) and patient samples (diagnostic performance). As the site for the trial, we selected Recife in the Pernambuco State of Brazil, which was the epicentre of the 2016 Zika virus epidemic in Latin America. As a mosquito-borne abrovirus (for example, dengue and chikungunya), viral spread in the region was exacerbated by the many peri-urban settings, which, due to standing water, can serve as breeding grounds for mosquitoes such as Aedes aegypti. Selection of Recife provided an opportunity to trial the diagnostic in a location of endemic disease and near, ultimately, where we envision local and distributed health centres could use the technology.
For this phase of the project, a standardized diagnostic workflow was used in all experiments where, after RNA extraction, samples were tested using Zika virus-specific NASBA and paper-based cell-free reactions ( Fig. 1b and Extended Data Fig. 1). The PLUM reader was used exclusively here and provided early and unbiased quantification of the colorimetric responses in real time. In parallel, as a gold-standard comparison, all samples were tested the same day using the RT-qPCR protocol for the Zika virus diagnosis, developed by the US CDC (cycle threshold (Ct) ≤ 38: positive and Ct > 38: negative) 25 .
To meet this need, we designed the PLUM reader, an affordable, computer vision-enabled, camera-based plate reader, which is essentially a 'lab in a box' that serves as a low-cost, temperature-controlled plate reader (~US$500; Fig. 2e). In PLUM, a low-cost camera and a credit-card-sized computer are used to collect and analyse the data from the entire multiwell plate in a single image capture. This strategy substantially reduces the complexity and cost of the device and moves much of the data collection burden to the software.
Device configuration. The PLUM reader was assembled (Fig. 2e) using a Raspberry Pi computer, off-the-shelf electronics and rapid prototyping techniques such as three-dimensional (3D) printing and a laser-cut acrylic housing. Much like a conventional plate reader, users interact with a user-friendly graphical user interface (GUI) ( Fig. 2f and Supplementary Fig. 1) that coordinates all functions of the PLUM reader. An LCD touch screen is integrated into the device for a simple and compact system that can be easily brought, as we did for this project, to multiple sites of use. This toaster-size device (20 cm × 20 cm × 20 cm) weighs only 2 kg, which compares well to conventional devices that are considerably bulkier and can weigh as much as 35 kg (for example, BioTek Neo2). The device is capable, with the correct adaptor, of operating on global power supplies or can run for 8-9 h using a standard lithium-ion brick battery (100 Wh).
PLUM software controls the 3D-printed plate reader tray, powered by a servomotor, for automated operation and dependable control over tray position (Fig. 2e). Through the GUI, the user can setup the plate map (for example, control and samples wells) for onboard data analysis and plotting at the end of each experiment (Fig. 2f,g and Supplementary Fig. 1b-d). Once the plate is loaded, the position of the plate is identified through a validation algorithm that automatically finds reusable yellow acrylic markers that sit in the four corner wells of the plate for image alignment ( Supplementary  Fig. 1a). Upon recognition of these markers, a digital template of the multiwell plate is used to assign the red, green and blue (RGB) channel pixel values for each well into regions of interest, which are used to create absorbance-equivalent data for all 384 wells. Collateral benefits of this approach are that PLUM's computer vision-enabled software can be easily configured to collect optical data from any plate format, and periodic calibration of device alignment is not necessary.
Light path. Another challenge to operationalizing the camera-based approach was the collection of high-quality, high-sensitivity data with such a simple hardware configuration. The light box, which is located at the top of the device, sits above the multiwell plate, with the camera looking up at the colour-based paper reactions in the plate much like an illuminated-stained glass window (Fig. 2e, Supplementary Note 2, and Methods for all design and component information). Our first attempts resulted in poor data quality due to high background signal from light passing through unused empty wells in the plate (data not shown). To resolve the issues of both low dynamic range and high variability over time, we applied commercially available opaque PCR foil to block incoming light from empty wells (estimated <US$1 per sheet) (Extended Data Fig. 1). This simple and low-cost solution has the added benefit of ensuring that the unused wells remain clean for future experiments.
With the basic optical system in place, we next optimized the quality of the light source in the device. Off-the-shelf white LEDs carry warm and soft undertones with low or high blue channel biases and do not provide the broad spectrum of wavelengths needed for a colour-based analysis method 31 . Accordingly, we sourced specialized broad-spectrum white LEDs, which are used for niche applications to simulate daylight, to provide a near-ideal coverage of the visible spectrum. The 54 LEDs (US$0.95 each) were arrayed using a custom-printed circuit board to create a simple and low-cost With the analytical specificity and sensitivity of the paper-based test established, we next set out to evaluate the diagnostic performance of the system using patient samples from suspected cases of arboviral infection collected during the 2015-2016 Zika virus outbreak in Pernambuco State, Brazil. To enable automated diagnostic analysis for users, we generated a logistic regression threshold using the time-resolved absorbance-equivalent readings from the sensitivity experiments conducted in the PLUM reader (Fig. 3c). This threshold (0.1603 arbitrary units (a.u.) above background) was then applied to the analysis of data from double-blinded, paper-based testing of extracted RNA from patient serum samples. The resulting PLUM-based analysis allowed for the differentiation of positive from negative samples as early as 130 min ( Supplementary Fig. 4c). A total of 268 patient samples were analysed using our diagnostic system in parallel with RT-qPCR ( Fig. 3d and Table 1). In comparison with the RT-qPCR assay 25 , we detected four false-negative and zero false-positive samples, which translates to a calculated 35 diagnostic accuracy and specificity of 98.51% (95% confidence interval (CI), 96.22-99.59 %) and 100%, respectively. Datasets from the field trial diagnostic (0.1603 a.u) and RT-qPCR (Ct ≤ 38) were plotted to show the similar accuracy of the respective thresholds in correctly segregating positive and negative patient samples (Fig. 3d).

Field trial of the diagnostic test for the chikungunya virus.
Given the success of the Zika virus patient trial onsite in Brazil, we sought to demonstrate the versatility of the PLUM reader with We began the characterization of the diagnostic system by testing the analytical specificity for the American strain of the Zika virus against a panel of seven endemic arboviruses that could, in practice, be found in patients with similar symptoms to those associated with the Zika virus. This included the chikungunya virus (CHIKV) and yellow fever virus (YFV), along with the four serotypes of the dengue virus (DENV-1-4). We also evaluated strain specificity with addition of the African strain of Zika (ZIKV Af). As can be seen by eye, a positive signal (purple colour) was only detected in the presence of the American strain of the Zika virus (Fig. 3a, bottom). The colorimetric response of reactions was quantified using the PLUM reader, with these results directly matching those from the parallel RT-qPCR assay (Fig. 3a, graph, and Supplementary Fig. 4a).
We next evaluated the analytical sensitivity of the paper-based Zika virus test. Here, we performed serial dilutions of the Zika virus after RNA extraction. The results demonstrate that the sensitivity of the paper-based diagnostic system is equivalent to the gold-standard RT-qPCR assay, with detection of the virus down to 10 1 PFU ml −1 (Fig. 3b and Supplementary Fig. 4b). The difference between the maximum sensitivity observed for synthetic RNA (10 0 RNA molecules per µl; Fig. 2b) and cultured virus (10 1 PFU ml −1 ; Fig. 3b) can be explained by the fact that the PFU metric accounts only for infective viral particles, whereas the diagnostic sensor can detect RNA from both infective and non-infective particles. Accordingly, it is not possible to directly correlate the results from synthetic RNA (Fig. 2b) to the lentiviral data (Fig. 2c,d) or viral stock (Fig. 3b).   Fig. 4b). NA, not applicable. Visual outputs at final time point of 235 min are shown below the graphs in a and b (yellow, negative; purple, positive). The graphs in a and b represent the mean ± s.d. of technical replicates from a single representative experiment (three independent biological triplicates were performed) at 130 min of the cell-free experiments (preceded by 70 min NASBA reaction). RT-qPCR experiments were run in parallel for results confirmation using the CDC gold-standard assay ( Supplementary Fig. 4a,b). c, Logistic test for threshold value determination (a.u.) of Zika virus diagnostic at 130 min, established using normalized background reading from analytical sensitivity tests performed in PLUM device. On the y axis, negative samples were plotted at 0; positive samples were plotted at 1. d, Segregation of the patient samples using the threshold value set in c is placed in perspective of the Ct values obtained by RT-qPCR for all 268 samples.
PLUM uses computer vision and an algorithm based on colour analysis, rather than optical absorbance, to provide functionality similar to conventional plate readers but at a fraction of the cost because of its simple design (Fig. 2e). The combined hardware and software of the unit provides a self-contained, automated and easy-to-use system and includes onboard data collection and analysis, incubation and cloud-based data storage.
These combined purpose-built molecular and hardware technologies come together to help move a proof-of-concept, lab-based assay towards providing low-cost, clinical-grade diagnostics at the point of need. Using 268 patient samples, we found that the paper-based Zika diagnostic could provide analytical specificity and sensitivity equivalent to RT-qPCR, with a diagnostic accuracy of 98.5% (Table 1). Similar performance for the chikungunya sensors shows promise for a path towards a generalizable diagnostic approach. The PLUM reader adds considerably to the molecular diagnostic assay by providing the option for portable use and, via signal thresholding, the automated discrimination of positive and negative samples. This translates to results as early as 2.5 h of reaction time (70 min NASBA + minimum 75 min cell-free reactions), which compares well with traditional RT-qPCR (1.5 h). Importantly, the potential for detection at the point of need could functionally reduce the time required for patient diagnosis from a scale of days to hours.
The Zika outbreak is over, yet the need for Zika virus diagnostics remains, and, according to a United Nations Development Program report, it is now expected that the Zika virus will become endemic in Latin America 14 . This prediction is supported by confirmed Zika cases in 2020, which, in Brazil alone, totalled approximately 7,000 individuals 43 . Transmission continues at low rates throughout the Americas and is currently also a concern in South Asia 44 . Although the spread of the virus is not currently an urgent concern, an estimated 2.6 billion people live in regions of the world that could establish local Zika transmission 45 , based on the presence of competent mosquito vectors and appropriate climate. By 2050, it is expected that almost half of the world's population will live in regions of arbovirus transmission as a result of Aedes aegypti spread due to urbanization and climate change 46 . Thus, the ongoing need for Zika and chikungunya testing, and the risk of future surges of infection, make these viruses excellent exemplars for point-ofneed diagnostics.
The PCR-free nature of our paper-based system also affords important benefits and has the potential to serve as a drop-in replacement for RT-qPCR in de-centralized applications. As we have seen with the coronavirus disease 2019 (COVID-19) crisis, the centralized nature of RT-qPCR can limit access to diagnostics and is prone to supply chain disruption during infection surges 47 . These same features, along with the challenges of cost and the logistics of shipping, also generally limit access to diagnostics in low-income and middle-income countries. Although not used here, our programmable gene-circuit-based diagnostics. For the next diagnostic demonstration, we chose the mosquito-borne chikungunya virus, which has symptoms overlapping with the Zika virus 11,36,37 . Like the Zika virus, the chikungunya virus originated in Africa and had recently arrived in Latin America, with a sustained and ongoing spread worldwide 38 . Symptoms include fever and soreness; and, although most patients recover within weeks, severe joint pain can last for months 39 . Chikungunya infections are similarly prevalent in peri-urban and low-income communities, and so, as for the Zika virus, there is a need for accessible and de-centralized testing 40 .
As with the Zika virus diagnostic 15 , we began with the computational design of 48 toehold switches targeting various regions of the chikungunya viral RNA genome. Each toehold switch candidate was linked to the LacZ reporter and tested for detection of the corresponding synthetic target RNA sequence (2 μM; Supplementary  Fig. 5). The top-performing sensor (number 10) was then optimized for analytical sensitivity using several combinations of NASBA primers specific to the region of the targeted chikungunya sequence. The combined NASBA and toehold switch-based test was able to detect the target synthetic RNA down to the clinically relevant range of 3.25 × 10 3 molecules per µl (5 fM; Fig. 4a) 38,41 .
With the molecular components of the diagnostic validated, they were distributed to the field site in Recife, Brazil. Here, the paper-based test was evaluated for diagnostic capacity using cultured chikungunya virus (strains from Paraiba-PB and Pernambuco-PE states) and tested for analytical specificity against a panel of eight off-target endemic arboviruses (including the related Mayaro virus (MAYV)). As before, the chikungunya diagnostic test provided 100% analytical specificity for target strains (CHIKV PE, PB; Fig. 4b and Supplementary Fig. 6a). Similarly, when evaluated for analytical sensitivity using titrated chikungunya virus (strain PE), the paper-based assay provided detection down to 10 1 PFU ml −1 (Fig. 4c). Although the RT-qPCR assay 38 was more sensitive than the paper-based test ( Supplementary Fig. 6b), this did not seem to limit performance with patient samples. On the basis of the analytical sensitivity data of PLUM, a chikungunya-specific threshold value (0.08 a.u. above background) was established at 75 min (Fig. 4d). Diagnostic accuracy of the chikungunya test was compared to RT-qPCR (Fig. 4e) and calculated 35 to be 98.46% (95% CI, 91.72-99.96 %) in a double-blinded, 65-patient study, with only one false negative, and with the diagnostic sensitivity and specificity of 92.31% and 100%, respectively (Table 1, green, and Supplementary  Fig. 6c).

Discussion
This report provides the results of validation studies, onsite in Latin America, of paper-based synthetic gene networks 22,42 as diagnostic systems for the Zika and chikungunya viruses (Figs. 3 and 4). This work also extends the concept of low-cost and portable companion hardware by describing the development of the PLUM reader. For both ZIKV and CHIKV viruses, the diagnostic trial was performed using RNA extracted from patient serum. All data were collected and quantified using the PLUM reader. The patient trial data were collected over the course of 235 min. The segregation of positive and negative samples was performed at the 130 min time point for the ZIKV diagnostic using the set ZIKV threshold (Fig. 3c and Supplementary Fig. 4c) and at the 75 min time point for the CHIKV diagnostic using the set CHIKV threshold (Fig. 3d and Supplementary Fig. 6c). The 95% CIs are specified within brackets.
implementation of local and distributed reagent manufacturing (for example, NASBA and lysate-based, cell-free protein expression reactions) as crucial to realizing many of the logistic and economic advantages of distributed diagnostic tools. Work on this is already underway by others in the community, and proof-of-concept efforts show a clear path to ultra-low-cost diagnostic reagents [49][50][51] . Recent proof-of-concept work demonstrated onsite in Chile indicated the cost of producing the cell-free protein expression reaction (5 μl) to be US$0.069, which, here, would replace PURExpress and the related US$2.54 cost (Supplementary Table 2) 49 . Notably, access to diagnostics is not solely a consideration of cost. Reducing the burden of diagnostic testing by creating technologies that can be made using local inputs and expertise, and that are not capital intensive, will go a long way to improving public health equity in future pandemics. The development of low-burden techniques that can de-skill sample preparation and liquid handling steps for diagnostics is also key for the practical deployment of distributed diagnostics. With this in mind, here we have demonstrated two lab-based sample preparation methods. The first, sample boiling, introduces that possibility of essentially 'no-cost' viral lysis. Boiling has been established by others as an effective lysis method for diagnostics 52 , and, as we found for the engineered lentivirus (Fig. 2d), boiling was also an effective lysis method for the detection of cultured Zika virus (data not shown). The second method, column-based nucleic acid purification, was used for the extraction of patient RNA from samples.
paper-based technology has previously been shown to be amenable to freeze-drying, for distribution without refrigeration and in-house E. coli cell-lysate-based reactions, for low-cost testing 15,21,22 . We see emerging diagnostics, such as the paper-based tests presented here, as having substantial near-term potential to augment existing RT-qPCR capacity, improving equity in the access to health care and aiding the responses to public health crises.
Still, important technical challenges remain to be solved ahead of practical implementation. In this report, we have provided validation results for clinical-grade molecular diagnostics using gene-circuit-based sensors and equipment that are low cost and portable. However, as with RT-qPCR, these assays still require liquid handling by skilled users to prepare samples (RNA extraction of 30 samples in about 60 min) and assays. Although we did not experience issues of cross-contamination, the need for manual intervention will increase this risk as efforts move into less technical settings. Continuation of good laboratory practices will be important as technologies make this transition.
Other real-world challenges also remain to be met. This includes ensuring that the paper-based diagnostic technology is affordable. Our estimated cost per test is US$5.48 using the price of research-grade reagents (Supplementary Table 2); however, we anticipate that this cost will be further reduced at larger scales. This compares well to the price of RT-qPCR, which, at our site in Recife (Brazil), is US$11 per reaction 48 . Notably, we also see the   Supplementary Fig. 6b). NA, not applicable. Analysis in a-c represents the mean absorbance ± s.d. of technical replicates from a single representative experiment (three independent biological triplicates were performed) and represent the experimental time point at 75 min of the cell-free experiments (preceded by 70 min NASBA reaction). In some cases for a and b, data points of the replicates are below zero on the y axis. Statistical analysis of (b): unpaired t-test, ***P = 0.0007. RT-qPCR experiments were run in parallel of (b) and (c) for results validation ( Supplementary Fig. 6a,b).  NASBA reactions. Isothermal amplification of targets was performed using a commercial NASBA kit (Life Sciences Advanced Technologies NWK-1), following the manufacturer's instructions with slight modifications. In brief, reactions were performed in a 5 μl volume format. Per reaction, 1.67 μl reaction buffer, 0.83 μl of nucleotide mix, 0.05 μl of RNase inhibitors and 500 nM of each primer were mixed along with 1 μl of sample (water containing target RNA or sample lysate). Reactions were assembled at room temperature, incubated at 65 °C for 2 min and then at 41 °C for 10 min, before adding the enzyme mix (1.25 μl). This was followed by incubation at 41 °C for 1 h. Reactions were then added to the PURExpress reaction as previously described at a 1:7 dilution for detection of amplified target(s) 15 .
Cell-free reactions. Cell-free reactions (PURExpress, NEB, E6800L) were assembled following the manufacturer's instructions, as described previously 15  Cloning protocol. Trigger sequence corresponding to the Zika-virus-specific toehold switches was placed downstream of the mCherry sequence between the P2A and T2A sequences 57 . The final sequence was mCherry-P2A-T3T8-T2A with the plasmid named pUltra-hot-mCherry-T3T8 (at 5093-5384 bp from ORI).
HEK293T transfection with lentiviral constructs. Once at 70% confluency in a 10 cm plate, cells were transfected with polyethylenimine (PEI, 408727, Sigma-Aldrich) by combining a ratio of DNA:PEI at 1:3, following manufacturer's instructions and incubated overnight at 37 °C 58 . The ratio of plasmid DNA (µg) used was pUltra-hot-T3T8: pMD2.G: pMDLg/pRRE: pRSV-Rev = 4.5: 1.5: 3: 3. Media were then replaced with 10 ml of fresh culture media and incubated at 37 °C. Once mCherry expression was detected, media were collected and centrifuged at 1,000g for 5 min to pellet the debris and the supernatant was syringe filtered using a 0.4 µm filter (Millipore).
Lentivirus concentration. LentiX (TaKaRA, catalogue no. 631231, lot no. 1705016a) concentrating solution was added to the filtered supernatant in a 1:3 ml ratio (1 ml of LentiX to 3 ml of virus supernatant), and concentration was performed following manufacturer's instructions. Virus titration: HEK293T cells were plated at 3 × 10 4 cells per well. Serial dilution of lentivirus aliquot was performed in PBS, with 5-7 technical replicates for each, and 25 µl of the diluted virus was transferred onto the wells containing the cells. The plate was placed at 37 °C for overnight incubation.
Median tissue culture infectious dose. Plates were analysed for cytopathic effect, and conversion from median tissue culture infectious dose (TCID 50 ) to PFU was done using the calculation suggested by the American Type Culture Collection (https://www.atcc.org/support/technical-support/faqs/converting-tcid-50to-plaque-forming-units-pfu#:~:text=For%20any%20titer%20expressed %20as,mean%20number%20of%20PFU%2Fml). At our research site in Recife (Brazil), the cost per column is US$4.96, and so this should be considered in addition to the reagent cost described above (US$5.48; Supplementary Table 2). However, most current diagnostics on the market do not include the extraction step in their kit nor in their price.
Another emerging feature for point-of-need diagnostics is the incorporation of a control sensor specific to patient RNA (such as RNase P). This control serves to guard against false-negative diagnostic results by ensuring that diagnostic samples contain RNA of sufficient quality and quantity. Although we followed gold-standard RT-qPCR methods 25,38 at the time of our patient trial in 2019, a sample RNA control was not part of the standard practice and so was not included. False-negative results were not a significant factor in this work (Table 1); however, going forward, we envision that the implementation of such tools at the point of need will improve the utility and robustness of distributed diagnostics.
As synthetic biology moves increasingly towards the practical application of biotechnologies, this report also holds potential lessons for those seeking to validate other diagnostic systems 53 . As an early demonstration of gene-circuit-based diagnostics in the field, this work serves to inform the emerging low-cost diagnostics research community of the challenges of taking technologies from the bench and into practice in the field. This transition and the transfer of technology, as we and others have found, hold many challenges 54,55 . One main challenge was how to bring high-capacity optical characterization into new environments for distributed testing. We solved this with the development of the PLUM reader, and we anticipate that this hardware solution will help future efforts to validate and implement technologies in low-resource settings. There were also notable logistical challenges in bringing the technology from our laboratory to field sites. Accordingly, we have included brief notes that outline details that we found helpful (Supplementary Method 2). Studies that focus on the validation of technologies at the point of need will be increasingly important as the challenges of moving a technology beyond the proof-of-concept stage and towards a practical tool that can impact the world are tackled. Given the low projected cost and low technical burden for operation, we envision these and other similar technologies as a new generation of tools that will improve global access to needed diagnostics 17,56 .

Methods
Toehold switch and NASBA primer design method. Toehold switches were designed along with companion NASBA primers as described previously 15 . In brief, the target regions within the viral genome were analysed for suitable NASBA amplification sites based on primer sequence and thermodynamic characteristics and the secondary structure of resulting NASBA RNA amplicon. Toehold switches targeting the most promising RNA amplicons were designed and assessed based on multiple parameters, including the secondary structure of the toehold switch and the availability of the binding site in the amplified viral RNA. Combinations of toehold switches and NASBA primers predicted to provide the best performance were then selected for experimental screening.
Toehold switch construction. Toehold switches were designed and constructed using conventional molecular biology methods. Synthetic DNA templates (Integrated DNA Technologies (IDT)) were amplified by PCR using primers (Supplementary Table 1) and cloned into a pCOLA-Duet backbone in frame with the LacZ coding sequence using Gibson assembly, as described previously 15 .
Synthetic RNA target. DNA encoding the Zika virus target was obtained from the Collins lab in a pET15 backbone 15 . After PCR-based linearization of the template (New England Biolabs (NEB) Phusion, M0530L or NEB Q5 M0491L) using primers listed in Supplementary Table 1, in vitro T7 RNAP-based transcription of synthetic RNA was performed (NEB, E2040S). RNA samples treated with DNAse I (Thermo Fisher Scientific, K2981) were then purified (RNeasy Mini Kit, Qiagen, 74104) and used to perform toehold switch screening (2 μM RNA) or sensitivity assays (10 6 -10 0 molecules per µl). DNA encoding the target chikungunya sequence was ordered through IDT as dsDNA with a T7 RNAP promoter. The primers used to amplify the target DNA are listed in Supplementary Table 1. After amplification, in vitro T7 RNAP-based transcription was performed as described above and then purified.
The device contains a power jack that can be mated with a 12V, 7A power supply or a portable battery (TalentCell Rechargeable, 100 Wh) to support 8-9 h of operation in the field.
All of the information required for the assembly of the PLUM reader is supplied as Supplementary Information. Data files for laser cutting, 3D printing and circuit diagrams and the Gerber files required for printed circuit board manufacturing can be accessed at https://github.com/PardeeLab/ zikaproject_hardware.

Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. All the experimental raw data are available for research purposes from the corresponding author on reasonable request.

Code availability
Code for controlling the device and conducting experiments on the PLUM reader can be found at https://github.com/PardeeLab/zikaproject_plumcode.git. Additional code is available from the corresponding author on reasonable request. Virus strains, virus culture and purification. All arboviruses used in this study were provided by the Laboratory of Virology and Experimental Therapy, Oswaldo Cruz Foundation (FIOCRUZ), in Recife, Brazil.
Analytical sensitivity experiment. Viral RNA from stock of all viruses (at 10 6 PFU ml −1 ) was extracted using the QIAamp Viral Mini Kit (Qiagen, 52906) and eluted in 60 µl of water. After serial dilution of the extracted RNA (from 10 5 to 10 −3 PFU ml −1 ), samples were assayed in parallel with the gold-standard RT-qPCR and the NASBA/cell-free reactions 25,38 . ZIKV American strain (PE243) and CHIKV strain (PE2016-480) were the strains used in those studies.
Analytical specificity experiment. Analytical specificity of Zika virus or chikungunya virus was determined against a panel of different mosquito-borne viruses endemic in Latin America. Extracted viral RNAs of DENV-1-4, YFV, ZIKV American, ZIKV African, MAYV, CHIKV PE and CHIKV PB at 10 6 PFU ml −1 were used as inputs to NASBA/cell-free reactions and parallel RT-qPCR.
RT-qPCR for ZIKV and CHIKV. ZIKV and CHIKV RT-qPCR was performed according to protocols established by the CDC, with minor modifications 25,38 . Reactions were performed using the QuantiNova Probe RT-PCR kit (Qiagen) following the manufacturer's protocols for 10 μl of final volume with primers of both viruses in 800 nM final concentration and probes in 100 nM final concentration. Primers and probes for ZIKV and CHIKV can be found in Table 1. Reactions were carried out in a QuantiStudio 5 system (Applied BioSystems) with a thermal cycle programme consisting of a single cycle of reverse transcription for 15 min at 45 °C, followed by 5 min at 95 °C for reverse transcriptase inactivation and DNA polymerase activation, and then 45 cycles of 5 s at 95 °C and 45 s at 60 °C. All samples were tested in duplicates, with negative controls (all reagents except RNA) and positive controls (RNA extract from viral stock). For results analysis, the QuantStudio Design and Analysis Software version 1.5 was used with automatic threshold and baseline. Samples were considered positive with Ct ≤ 38.0. PLUM hardware. The PLUM reader consists of two chambers housed within a laser-cut acrylic box fastened together with metal screws and mounting brackets ( Fig. 2; McMaster-Carr, 8505K14 and 98164A061; Digi-Key, 36-621-ND). The light box is designed with 54 LEDs (YJ-VTC-5730-G01-65, YUJI LED) for illumination. The motorized tray is designed to hold a standard multiwell plate and positions the plate at the correct focal length for the camera (US$35, Raspberry Pi V2, 8-megapixel). This configuration allows for clear visualization of the plate from below for capture of the image data ( Supplementary Fig. 3 Fig. 1 | Graphical workflow. a, Reagent preparation: Linear DNA for toehold switches is obtained by PCR from a plasmid vector. Similarly, positive control trigger DNA is obtained from plasmids via PCR and subsequently in vitro transcribed into RNA. Column purification steps are indicated. b, Material preparation: Yellow acrylic markers are laser-cut to fit into the four corners of a 384-well plate. Paper discs are punched out from BSA-treated filter paper using a 2 mm biopsy punch and deposited in the bottom of each reaction well. The wells surrounding the reaction are then filled with water and an aluminum foil seal is placed on the plate. Using an utility knife, only the reaction wells are cut out. c, Diagnostic assay: Viral RNA is extracted from patient samples and then amplified through NASBA isothermal amplification, alongside positive control trigger RNA. The amplification product from NASBA is then added to cell-free reactions with linear switch DNA. Clear PCR film is applied to the plate to seal the reactions. The plate is placed inside the pre-incubated PLUM device. d, Reaction analysis and data collection: The PLUM software recognizes the plate and continues to capture the reaction (5 min intervals) over the next three hours. At the end of the reaction, the software analyzes the images generated, determining whether a purple to yellow color change has occurred in each well. PLUM generates a graphical report, which indicates whether the sample is positive or negative using a pre-determined threshold value.
Corresponding author(s): Keith Pardee Last updated by author(s): Dec 23, 2021 Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection The experimental data were collected using custom code, available on Github at https://github.com/PardeeLab/zikaproject_plumcode.git.

Data analysis
The logistic test was performed using STATA 15. The accuracy test was performed using https://www.medcalc.org/calc/diagnostic_test.php. Data analyses on the PLUM reader were performed using custom code available at https://github.com/PardeeLab/zikaproject_plumcode.git.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The main data supporting the results in this study are available within the paper and its Supplementary Information. All the experimental raw data are available for research purposes from the corresponding author on reasonable request.