Integrated & Gold Human Practices

Introduction

Through targeted practices, we were able to gain thorough insights for each specific aspect of our project as it evolved. The flowchart summarises the integration of our human practices during the whole project. We chose to prioritise our contacts to address information not covered by the published literature, obtain the most up to date and relevant facts, and develop a understanding of the future directions of our project. We also aimed to maintain as sustained dialogue with the same experts as our ideas progressed during the project, to help us obtain more specific and useful insights that are outlined below.

Wet Lab

Professor Maike Bublitz

Professor Bublitz of the University of Oxford researches the molecular mechanisms of protein transport. We contacted her to obtain advice on the optimisation of our protein purification protocols and designing in vitro TEV protease assays to more accurately represent our cell free system was invaluable to our wet lab. Unfortunately, due to complications in the wet lab we were unable to purify the required proteins the first time round and could not repeat the protocols because of time constraints. However, her advice remains highly relevant for the characterisation of our parts in a cell-free environment, which is something we hope to pick up on in the future.

Dr Miguel Hernan Vicco

Dr Vicco is a researcher at the Universidad Nacional del Litoral in Argentina currently working on immuno-endocrine parameters marking the course of Chagas disease, and on vaccine development against Trypanosoma cruzi. We approached him for information about cruzipain as a biomarker. He explained the potential difficulties of low specificity, sensitivity and undefined concentration associated with using cruzipain as an antigenic biomarker in his work. However, since our project involves the detection of the protease activity of cruzipain, the usual problems of a low antigenic specificity and sensitivity when using cruzipain as a biomarker are overcome for the protease activity of cruzipain is specific. Nonetheless, the insight provided by Dr. Vicco on the uncertainty of the concentration of cruzipain in the blood further reinforced the modelling results to indicate the need for an integrated amplification system in our biosensor.

Professor Emilio Malchiodi

Professor Malchiodi is a researcher at the University of Buenos Aires developing vaccines for Leishmania and Trypanosoma cruzi. Our discussion with him highlighted the issue of variable cruzipain concentration in the acute phase with the lower range likely only resolvable by sensitive techniques such as Surface Plasmon Resonance (SPR). Since we lacked the resources for SPR and did not have access to parasite cultures, due to safety reasons, it also emphasized the need for amplification in system to allow detection of the lowest possible concentrations of cruzipain. In the future, we hope our diagnostic test can be trialled in mouse models with naturally variable levels of cruzipain to confirm the viability of the amplification system in detecting the lowest levels of parasitemia in the acute phase of Chagas disease from one or less than one parasite in the sample.

Dry Lab

Dr Scott L Diamond

Dr Diamond of Penn University is an expert in blood systems biology and protease proteomics, who we contacted for advice on the accuracy of parameters used in our blood clotting model. He informed us that 10 nM thrombin is necessary for blood clotting, allowing us to improve our models by increasing their accuracy. More accurate models consequently enabled us to determine the properties of our DNA and protein-based circuitry with a greater precision, and refine their design to optimise the detection of cruzipain and lower the chance of false positives and false negatives in our test.

Professor Mike Laffan

Professor Mike Laffan, professor of Haemostasis and Thrombosis at Imperial, advised us on the kinetics of blood clotting. He brought to our attention that the release of hirudin during the blood clotting cascade successful output results in a race between the two unpredictable processes. Therefore, he recommended the use of citrate anticoagulant to delay blood clotting, giving sufficient time for hirudin release, before addition of calcium ions and a coagulation trigger such as a tissue factor. We consequently decided to add these parameters to our model and adapt our kit design by adding sodium citrate freeze dried with our DNA in the in well and calcium and the trigger factor in the capillary coating inlet.

Professor Matt Higgins

Professor Matt Higgins is based at the University of Oxford and works on interactions that human-infective parasites make with their human host, focusing on the parasites that cause malaria (Plasmodium falciparum) and sleeping sickness (Trypanosoma brucei). He uses structural and biophysical methods to characterize these host-parasite interactions, and to guide the development of improved vaccine components. We initially approached him to learn more about the detection of Trypanosomes within the human host but he informed us that the differences between the lifecycle of T.brucei and T.cruzi were too different to be able to draw direct parallels. However, we were able to use the insight he provided into other pathogens to improve the potential of our project being used as a platform technology, by swapping the input and outputs as required. More information about using the use of our diagnostic as a platform technology can be found along with our software tool here.

Professor Mike Bonsall

Professor Mike Bonsall is a Professor of Mathematical Biology in the Department of Zoology at the University of Oxford. He is a population biologist and has research interests across a range of disciplines including biodiversity, ecology, evolution, health and economics. We approached Mike to gain a better understanding of the principles of disease modelling, and equip us with the skills to create our own epidemiological model for Chagas disease that illustrates the impact our device would have in a case study scenario, based in Bolivia. The disease model helped evaluate the validity of our real world problem, confirming that screening of all infants through a rapid point of care diagnostic would be impactful. More information regarding our disease model can be found at the following the link.

Professor Mark Howarth

Professor Howarth is researcher at the University of Oxford working on protein nanotechnologies for cancer analysis and immune activation, and designer of the spytag-spycatcher system for bioconjugation. He provided information on the kinetics of the spytag-spycatcher interaction that was invaluable to our modelling of the incorporation of our constructs into OMVS. Further information on the OMV models can be found at the following the link.

Chagas Disease

Professor Yves Carlier

Professor Carlier is a researcher in infectious diseases and clinical immunology at the Université Libre de Bruxelles. We first contacted him early on in our project to gain more information about the pathology of congenital Chagas disease. He informed us about the benefits of diagnosing Chagas disease in neonates, such as that treatment with benznidazole cures around 100% of babies if given before one year of age but not later in life. He emphasised the need for a diagnostic for neonates that would detect alive parasites, strengthening our resolve to focus on congenital Chagas disease. To ensure that our test would be triggered only by an active infection of living parasites, we decided to design the diagnostic so it would detect the circulating cruzipain naturally released by living parasites into the blood and would not involve the lysis of the parasite to release cruzipain.

Dr. Carlier also directed us towards specific reading, such as his latest review on Maternal-fetal transmission of Trypanosoma published in 2017. Information from his review and other published research helped us estimate the parasite concentration in the blood of newborns and immunity parameters after treatment, which were required for our modelling, and about which very little is known due to a lack of long term studies. Further correspondence with Dr. Carlier provided us with insights into the parasitology of congenital Chagas disease influenced our applied design. For example, the examination of biological samples of the same neonates weeks after birth increases the sensitivity of parasitological detection since the neonatal parasitic loads often increase up to 13 months after delivery, consequently we recommend testing all infants using our kit twice in the optimal diagnostic strategy for congenital chagas disease using a rapid protease detecting kit.

Dr Cristina Alonso-Vega

Dr Alonso-Vega of the University of San Simón, Cochabamba who ran the National Congenital Chagas Disease Programme in Bolivia from 2004-2009, and is an expert in congenital Chagas gave us specific guidance for implementation of our diagnostic device in Bolivia. She confirmed the suitability of our congenital test in Bolivian hospitals, where most deliveries occur. We also learnt that Chagas treatment for under 15’s is free, giving us confidence that our diagnostic would be accessible and impactful in containing Chagas if provided in a hospital setting.

Our discussion with her further emphasised the requirement for a rapid point of care test that would allow treatment of infected newborns to begin before they left the hospital, which led us to re-evaluate the desired output of our kinetic models. As Dr. Alonso-Vega explained, the need for such a test stems from lack of medical follow-up and recommended we align the second test suggested in our optimal diagnostic strategy with the pentavalent vaccine to increase turnout of infants tested to further minimize issues caused by the lack of adequate follow up. The optimal location to obtain the newborn’s blood required for our diagnostic kit was also confirmed with advice from Dr Alonso-Vega. She suggested we consider placental and heel prick blood as two potential options as the blood source. Further research indicated that despite the high volume placental blood (10-20ml at delivery) it may not be as suitable in the context of our blood clotting reporter, because the blood from the placenta is likely to have already clotted. Thus we chose to opt for a heel prick based test, corroborating our survey results that 97% of those surveyed would be comfortable with a pinprick or small syringe test for diagnosis.

Eileen Murphy

We approached Eileen Murphy and colleagues, who led the PATH diagnostics gap analysis for Chagas, to help us contextualise our device in the existing diagnostic landscape and ensure our device was focused on meeting the required needs. Our communication with those at PATH reaffirmed our diagnostic focus on congenital Chagas disease, because outside of infants born to Chagas-confirmed mothers, the acute phase is typically mild, relatively short in duration meaning that many patients do not routinely seek medical care. In addition we were warned of the technical barrier of true negatives facing commercial assays detecting parasite infection directly, which occur due to intermittent shedding during chronic phase infection that means parasites may not always be present in collected blood samples. However, it was noted that this may not be as significant an issue if our diagnostic was used in the acute phase context. In light of these discussions we decided to tailor our device towards diagnosing congenital infection in newborns.

Professor Jaila Dias Borges Lalwani

Professor Dias Borges Lalwani is professor of epidemiology at the Faculty of pharmaceutical sciences at UFAM, whose contact details were forwarded to us by the AQA Amazonas team. We were fortunate enough to be able to arrange a Skype meeting with Jaila, which helped us contextualise the problem of Chagas highlighting the difficulty of seeking diagnosis given non-specific early disease symptoms. Furthermore, our conversation highlighted the regional specificity of transmission, directing us towards Bolivia as our focus country since it has the highest rates of congenital Chagas. We were also able to question Jalia about potential concerns highlighted through our meetings with HeLEX, such as the stigmatisation of Chagas disease in Latin America. An insight into the public opinion on Chagas disease was invaluable in designing our Chagas public policy that can be found by following the link below:

Applied Design

Dr Carol Harris and Dr David Harris

Dr Carol Harris advised us on the practical and social considerations involved in the application of our diagnostic in Latin America, based on her experience working in Paraguay. We discussed the current tests available for Chagas and Carol emphasised that a spot test would be by far the best option for a multitude of reasons. We then took this forward as a key criteria for our device. Carol spoke to us about the necessity for a clear output system that would cater to all education levels and not require specialist training to interpret, as this could limit its use in some places. This reaffirmed our idea of using blood clotting as an output in our device which will have a clear unsubjective positive/negative result. Following these discussions, we spoke about the practicalities of obtaining a blood sample for our diagnostic. Carol mentioned that finding professionals who can take blood could be difficult to find at all times, so a finger prick test would be better. This corroborated our previous research and information from other experts in this area. Our modelling later confirmed a finger prick test would be feasible even with the lowest concentration of cruzipain.

Tim Ring

Tim Ring, is the vice president of safe-tec sales and marketing and generously sent us MICROSAFE® pipette samples so we could test the compatibility of these with our kit design. These pipettes are advantageous for blood collection, as they eliminate the requirement for lining the collection tube with anticoagulant since they use a medical grade thermoplastic shown to delay FACTOR XII of the coagulation cascade. Using the samples provided by Tim allowed us to test our applied design more rigorously, and obtain information such as the the time taken from blood collection to delivery to the assay chamber being only 30 seconds.

Sarah Dragonetti and Dr Ben Riley

Sarah is a registered nurse who we contacted to evaluate our 3D printed kit prototype. She liked the good fit of the flat pipette hole, intuitive size and shape, and timestrip which would be a useful tool during busy periods. However she recommended the use of a window so the emptying of the pipette could be seen to prevent accidental redrawing of blood, and suggested a translucent casing in place of a red casing to better see blood through the window. She felt it was unclear how the components clicked together or whether the pipette should be separate or contained within the kit. General practitioner, Dr Ben Riley, liked the sealed kit for blood containment, small size for ease of packaging and transport and distinctive shape for ease of identification. He also suggest a target for the cost of the kit as £3, comparable to a one-use test diabetes strip. The insights from Sarah and Dr. Riley were hugely useful in assessing and refining the practicality of our diagnostic kit, consequently improving our applied design.

David Sprent, Juan Solano and Alfons Van Woerkom

David Sprent, an expert in International Supply Chain, and Juan Solano and Alfons Van Woerkom, representatives of the Global Fund, advised us on evaluating costs of manufacture, transport and taxation of a kit for end use by the Bolivian healthcare system. Their help was invaluable in cost analysis of our kit for the applied design section.

HeLEX and InSIS

We consulted with Dr Michael Morrison from HeLEX, which is the Centre for Health, Law and Emerging Technologies and specialises in investigating the relationships between law, ethics and practice in the area of emerging technologies in health. We also worked closely with Dr Louise Bezuidenhout and Dr Chris Goldsworthy at InSIS, the Institute for Science, Innovation and Society at the University of Oxford. InSIS researches and informs key contemporary and emerging issues and processes of social, scientific, and technological change. The insights from both HeLEX and InSIS were highly useful in evaluating the ethical and social issues related to our project, and heavily influenced our applied design. For more information see our Silver Human Practices page.

Professor Keith Pardee

Professor Keith Pardee is an assistant professor at the leslie Dan Faculty of Pharmacy, University of Toronto. His research looks into the potential of moving synthetic biology outside of the cell, rather than using cells to host engineered genetic programs. Having read about Keith’s research, some of our team attended one of his talks on cell-free technologies. He informed us about how microorganisms are not compatible with a lot of applications of synthetic biology, and how cell-free systems could solve a lot of these issues. This was our first big introduction to cell free and we became very excited about the possibilities this would have for our project. His talk also highlighted the key future goals of biotechnology as being low cost, using simple technology, and ensuring the difficult science is completed before application - which can be achieved by cell free technologies. Furthermore, cell free systems do not require a cold chain to be maintained during transport and storage making them ideal for our diagnostic destined for use in the field. In addition, Keith suggested that 37 degrees is the optimum temperature therefore our device would still function in Chagas endemic regions which have a high average temperature.

After the talk we approached Dr. Pardee for project-specific advice, including practical advice on the sequence of addition of certain components of our system such as adding TetR before freeze-drying to obtain greater activity. He pointed us towards a protocol for making our own cell-free extract. He further suggested that YFP on a paper system may be problematic as the auto-fluorescence of the paper may match the YFP signal, however this is no longer an issue as we later chose not to use a paper based system for our diagnostic. Dr. Pardee’s suggestions for future experiments, we could do with blood were promising especially as he said he had tried to get a cell-free system to work in blood plasma, giving us the confidence to continue with the idea of being able to use a blood sample as our detection medium. Finally, he also gave us some advice on how long this cell-free system would take to get started, which we were able to input into our models and gain a more representative values for our parametres.

Dr Piers Millet

Dr Piers Millett is a Senior Research Fellow at the Future of Humanity Institute, where he focuses on pandemic and deliberate disease and the implications of biotechnology. In our meeting with him, we discussed the policy brief that we were drafting at the time. He corroborated what Dr Lousie Bezuidenhout had told us about the importance of data management. We discussed the different ideas around regulation, such as the GMO vs ‘fast-evolution?’ debate. We also talked about our cell free report, for which Piers gave us his expert opinion on the current direction that regulation may be moving in; for example product based regulation over process based evolution. The cell free report can be viewed below:

From the meeting with Dr. Millet, we came away thinking about some of the key questions that we would have to ask ourselves about our project. Could we ever say that our device is ‘safe’? Or is it just safe enough, considering our current knowledge?. This impacted our final design by making us conscious of the potential barriers we could face in this new area of synthetic biology. For example, our device is within a contained cassette based on advice that a closed system would make it safer to use and easier to dispose. Piers also introduced us to the concept of platform technologies and how this could be applied to our project. Having a platform provided a good way of fast tracking future developments because the platform will already have met regulatory approval, therefore allowing the device to be more rapidly adapted for other diseases.