Integrated Human Practices
During the whole course of the iGEM competition, we tried to keep an open dialogue with experts, as well as with the society. Our ultimate goal was to keep our project aligned to the real societal needs around MERS-CoV detection and rapid containment during a potential outbreak. The received feedback let us understand the real challenges behind field diagnostics and enabled us to broaden the existing diagnostic approaches to novel paths. By doing so, we hope that we set a solid basis which can actively help and inspire researchers and healthcare practitioners all over the world.
The Hellenic Pasteur Institute
During the brainstorming phase of the competition, before concluding with our project, we had the chance to discuss with different researchers from various backgrounds. Our visit to the Hellenic Pasteur Institute during these very first steps introduced us to the fields of travel medicine and diagnostics. We understood the significant difficulties that viral detection and monitoring enclose. Dr Pelagia Foka highlighted the necessity of alternative on-field detection methods for the prevention and containment of future epidemics caused by the Middle East respiratory syndrome coronavirus (MERS-CoV). The need for portable diagnostic alternatives, detached from intensive laborious techniques posed a specific scope for the rest of the project.
After discussions with Dr Petros Iliadis, we realized the constraints of classic immunoassays currently in use for disease diagnosis, such as the protein production and purification cost that sometimes make this kind of tests inaccessible to the end-users. In addition, since these tests detect the products of the immune response against the parasite, they cannot detect the infection in early stages, before the response occurs (window-period). For the aforementioned reasons, we turned our attention on the very promising field of molecular diagnostics for viral detection.
Setting as our primary goal the development of a molecular detection method suitable for on-field diagnosis, we yet had to figure out how to get there. Harnessing the possibilities of synthetic biology, we decided to make use of the toehold-switch technology in a cell-free environment in order to detect the MERS-CoV viral RNA. This journey would have been impossible without the invaluable help of the personnel of the Molecular Virology Laboratory of the Hellenic Pasteur Institute and especially the contribution of Dr. Pellagia Foka who served as our instructor.
Dr Lee Alissandratos, Australian National University
Toehold-switches have previously been exploited for the detection of viruses. Research conducted so far has focused on correlating a change in colour or fluorescence to the presence of specific RNA sequences in a sample. Our initial plan was to design toeholds which would regulate expression of a downstream gene encoding GFP or HRP, correlating toehold unfolding to a change in fluorescence or colour respectively. However, the interpretation of an optical signal often requires further processing, via special lenses or mobile applications, e.g. through the use of a smartphone camera. Although Mobile Diagnostics is a promising sector for field applications, they are far from being standardized.
Dr Lee Alissandratos from The Australian National University provided his feedback on numerous occasions throughout the course of our project. As we were in search for “smart” reporter proteins that could facilitate the function of our proposed mechanism, Dr Alissandratos suggested coupling our virus detection mechanism with a robust and standardised low-cost technique, widely available and utilised at PoC: glucose monitoring with strips. Toward this aim, we concluded in designing toeholds regulating the expression of a gene that encodes trehalase; an enzyme that hydrolyses the glucose-dimer trehalose, leading to glucose accumulation when present in the sample. The glucose final concentration can be measured with a simple off-the-self glucometer thus enabling diagnosis.
Service to the Society
Mr. Panagiotis Doukas, Microbiologist, Analysis Medical Laboratories
The instructions concerning MERS-CoV diagnosis suggest sampling from the pharynx or the lower respiratory system. According to literature, the viral load in the sputum is slightly higher than that of the pharyngeal smear. However, we had concerns on which sampling method is more friendly to the patient and would be more suitable for a POC diagnostic kit. On one hand, not everyone is familiar with spit but on the other hand, the oropharyngeal sampling needs to be performed with the assistance of a second party. We reached out to Dr Doukas and he shared with us his insight concerning different sampling methods. He informed us that we could facilitate the secretion of sputum giving the patient an expectorant. Finally, as discussed below, we concluded to the oropharyngeal sampling, as it is also suitable for infants and the sample is easier to handle.
Hellenic Society of Point-of-Care Testing
We also had the unique opportunity to meet with Dr Dionisis Vourtsis, Dr Giorgos Antonakos and Dr Katerina Stini from the Hellenic Society of Point-of-Care Testing. They gave us general guidance as to the established criteria in order for a kit to be considered suitable for PoC testing and helped us define our primary goals for the kit design. They specifically noted the need for rapidness, precisely the diagnosis should be completed in 30-60mins, and automation. Taking this advice into account, we opted for RPA as a quicker RNA amplification method, instead of our original choice of NASBA. In parallel, we tried to combine several steps together, reducing the moves and the time needed for the test operation. This meeting also finalized our choice on the sample type, as we knew that the viral load is greater in the sputum than in the pharyngeal smear, but we did not know which sampling method would be less inconvenient for the patient. We were informed that patients are in general more familiar with the swab as a sample collecting method rather than the spit. Evaluating the difference in the viral load versus the ease in the collecting and handling of the two samples, we concluded to the collection of pharyngeal smear with a swab.
Dr. Maria-Nefeli Tsaloglou, Diagnostics For All (DfA)
Our kit design was extensively based on a diagnostic device developed by researchers at DfA. Dr. Tsaloglou, board member of the not-for-profit company, was eager to show us the device and answer our questions. Having worked with various amplification methods, she helped us pick a suitable one for our system. We had in mind that in order to avoid the need for cooling mechanisms and ensure a uniform temperature throughout the detection process, we should use an isothermal amplification method functioning at around 37
oC, that is the working temperature of our cell-free transcription-translation system. Dr. Tsaloglou indicated the disadvantages of NASBA and RCA, that we originally had in mind, such as being thermally initiated at 37-40
oC and also requiring a first heating step at 65
oC, that if omitted, would probably hinder the quantification. Instead, she suggested using RPA, that is chemically initiated with the presence of MgOAc and also functions at low temperatures (37-42
oC). Dr. Tsaloglou also gave us an insight into the scale of a reasonable cost for such a device, targeting low-resource POC facilities. She recommended that, in the end, we should perform a cost-of-goods analysis and take into account the upscaling for calculating the final cost of our kit.
Professor Aashish Priye, University of Cincinnati
Aashish Priye, Assistant Professor in the University of Cincinnati, is an expert on Lab-on-a-Chip applications in diagnostics and has worked on the development of a platform for rapid detection of viruses, such as Zika. We had the chance to discuss with him in depth some aspects of our kit design. To begin with, we presented to him our idea for the reagents' storage in a bubble-like structure that would break with a plastic needle letting the liquids to be absorbed by the paper disc. He underlined that given the volumes needed, this method of storage would be very convenient as it would prevent evaporation. Professor Priye also emphasized that the reagents encapsulation process should be uniform so as to be precise on the contained volume, and suggested the use of a self-sealing polymer to facilitate the process.
Professor Keith Pardee, University of Toronto
Professor Pardee's work has inspired us in many different stages of our project. We had the honor to discuss with him a few features of our diagnostic kit. Regarding the RNA extraction, in the beginning, we were thinking of including a heating of the sample at 95oC, as it is commonly done in laboratories. However, Prof Pardee suggested that we should start with the simplest approach and use an RNA extraction kit in our design proposal. In this way we can avoid excess energy needs, as well as, minimize the insulating materials cost. He also advised us to keep the RNA amplification and the cell-free transcription-translation system in separate steps, since there are indications that this way the two systems show better performance. Finally, he gave us some useful tips concerning the fabrication of the paper discs and the reagents quantities.
Dr. Robert J Meagher, Sandia National Laboratories
Dr. Meagher has worked in the field of microfluidics in POC diagnostics. He was eager to read our kit design proposal and offer valuable feedback. He clarified several aspects on the heating of the kit, such as the need to control the temperature in order to avoid overshoots and oscillations that might affect our mechanism. Dr. Meagher also suggested that a simple custom-made heat element from ceramic resistors, combined with a piece of aluminum to spread the heat, would be efficient and cost-effective.
He also offered us his view on the reusability of the kit, advising us on designing half the kit reusable and half disposable: the heater and the electronics will be reused and everything that comes in contact with the sample will be discarded, to avoid carryover contamination.
Future perspectives: Our kit as a generalized platform to assist viral detection
The detection mechanism we have chosen to implement for the detection of MERS-CoV is highly flexible, as it can also be applied to the diagnosis of other diseases. Toehold switches can be designed to respond to any RNA sequence; applications of this technology in Ebola and Zika diagnosis  have already been published. Toehold switches could even be used to bind and sense microRNA molecules that are characteristic of cancer or metabolic disorders .
In our effort to explore the potential of toehold switches and use them to good advantage, we thought of developing a similar molecular test for the detection of West Nile virus (WNV). According to the European Center for Disease Prevention and Control, a threefold increase in human cases of WNV infections has been reported this year, with 1266 cases in European Union Member States, 261 of which in our country, Greece.
To evaluate these ideas, we met with Helen Giamarellou, Professor of Internal Medicine at the Athens University Medical School and infectious diseases specialist currently working at the Hygeia Hospital, and discussed the application of this mechanism on MERS-CoV and other possible viruses. Regarding MERS-CoV, Prof Giamarellou confirmed the need for rapid mass testing in POC facilities and airports, so as to immediately diagnose international travelers that show symptoms of infection. However, she had reservations about the use of toehold switches in the detection of WNV. This virus does not remain inside the host cells for long periods and therefore, it is generally recommended to detect the antibodies and not the viral RNA.
On the other hand, Prof Giamarellou expressed particular interest in the application of this diagnostic method in influenza, a virus that shows high mutation rates and frequent genetic reassortment. This would facilitate the identification of new strains, yet high mutation rates imply low percentage of conserved regions, on which toehold switches are based, rendering the determination of suitable switches challenging.
 Pardee, K. et al. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell,165(5), 1255-1266.
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