Team:Oxford/HP/Gold Integrated
Integrated and Gold Criterion Human Practices
Dialogue with experts, stakeholders and industry informed our decision to focus on acute congenital Chagas disease
Further research focused us on neglected tropical diseases (NTD) because this is where we felt our diagnostic would have most impact (FINDdx, 2015). Chagas disease was chosen after identification of the cruzipain protease as a potential unexplored disease biomarker. Many other parasitic diseases use specific proteases in their infection mechanism giving scope for the development of an adaptable modular diagnostic device with common input and output modules by variable cleavage sequence.
However, literature search and communication with experts researching the use of cruzipain as a drug target exposed the difficulty of quantifying a cruzipain blood concentration due to variable parasite concentration (Emilio Malchiodi, personal communication) making it difficult to ascertain our expected output through modelling. In response to this we designed a split protease based amplification system to increase sensitivity of our detector circuitry.
A PLOS analysis (Albert Picado, 2017) identified a point of care test for congenital Chagas disease as the number one need in Latin America and priority for the world health organisation and ministry of health, despite being lower priority for researchers, therefore we decided to tailor our design to a congenital diagnostic to meet this unmet need (Table 1). Currently there is a 10 month wait for children born to infected mothers for diagnosis, missing the critical window for detection when parasite concentrations are highest a few weeks after birth due to the unsuitability of antibody based rapid diagnostic tests and lack of medical infrastructure (PATH, 2016).
| Problem | Our Solution |
|---|---|
| Lack of medical infrastructure in the worst affected areas compromising follow up and results distribution | A rapid point of care test optimised through modelling |
| Antibody based tests cannot be used due to presence of maternal antibodies | A non-antibody based test |
| Blood smear analysis requires training, and PCR tests specialised equipment | A simple test that does not require specialised equipment or medical training using blood clotting as a visible output |
Table 1: Evaluation of key problems producing demand for a congenital diagnostic and how our kit seeks to solve these
Expert Contact
Guidance for implementation of our congenital diagnostic device in Bolivia
As emphasised on our silver page meeting with epidemiology professor Jaila Borges from UFAM Brazil helped us contextualise our project, and directed us towards Bolivia as the country with the highest rates of congenital Chagas.
In light of the high incidence of congenital Chagas in Bolivia we chose to further investigate the specifics of implementing our device here. Our communication with Dr Cristina Alonso-Vega confirmed the suitability of our congenital test in Bolivian hospitals, where most deliveries occur. Furthermore, we found out that the Bolivian government guarantees Chagas treatment for under 15’s and provides free national insurance to mothers and children, giving us confidence that our diagnostic would be accessible and impactful in containing Chagas if provided in a hospital setting.
Image 1: Chun and Helen skyping Dr Alonso-Vega whose advice was incredibly helpful in development of a kit to meet the specific needs of the Bolivian healthcare system
The discussion also emphasised the requirement for a rapid point of care test, leading us to reevaluate the desired output of our kinetic models (link to modelling page) . As Dr Alonso-Vega explained, the need for such a test stems from lack of medical follow-up. Therefore, although in 70-80% of cases women are serology tested for Chagas and newborns born to Chagas positive mothers diagnosed by the microhematocrit, if these fail follow controls 2 months and 8-12 months after are necessary but inconsistent.
Current serology tests takes 10-15 minutes, and often much longer in the context of a busy hospital. It is not uncommon for results from laboratory verification often to come back after the mother has left. (Dr Alonso-Vega, personal communication).
The issue of blood source for our kit was also confirmed with advice from Dr Alonso-Vega. Placental and heel prick blood were considered as two potential options as the blood source, with the placenta giving 10-20ml blood at delivery. However, 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 blood is likely to have already clotted. Furthermore the heel prick option corroborates the results from our survey to Latin American teams (link to silver practices page) that 97% of those surveyed would be comfortable with a pinprick or small syringe test.
Use of cell free technology
We also Freeze-dried cell free systems are low cost, portable and do not require specialised lab equipment (Pardee K, 2016) making them ideal for a field diagnostic. We developed our understanding of the current progress in cell free systems by attending the OpenPlant forum in Cambridge where we were lucky enough to gain advice about making our system cell free from experts, especially Keith Pardee and Simon Moore. We were advised to optimise our output by adding TetR instead of expressing it in our system and were recommended protocols for obtaining cell free lysate and advised on commercial cell free expression kits.
Image 2: John and Noah with Professor Keith Pardee, whose papers inspired our cell free DNA-based system
Image 3: Noah and John with Tom Knight, synthetic biology pioneer and all-around wizard
Policy and Ethical Issues
Country specific regulations must be considered in implementation and dissemination of a diagnostic kit. To address these issues we approached Louise Bezuidenhout from HeLEX who advised us in helping us develop a policy proposal in the Challenges surrounding synthetic biology diagnostics in developing countries which can be read here.
We were also grateful to receive safety advice from Piers Millet on safety of cell free technologies, further detail of which can be found on the safety page.
