Team:Thessaly/Description
DESCRIPTION.
Refugees and migrants are in a very vulnerable position, deprived of their fundamental human rights, including access to healthcare. Tuberculosis is a leading health threat for populations affected by crises who lack access to innovative diagnostic tools. Taking these into account, we decided to develop ODYSSEE, a modular platform for field diagnosis of Tuberculosis aimed at being applied in refugee camps in our country as well as worldwide.
From the very beginning of our brainstorming sessions, humanitarian issues and their impact on everyday life have sparked our interest. During our conversation with one of our member’s friend, Emmanouela Daka, who is occupied as a social worker at the Reception and Identification Center (RIC) of Lesvos, she enlightened us about the living and healthcare conditions existing there. The observed limited access to health provisions led us to decide to use the principles of Synthetic Biology as a means to take a step towards achieving universal health coverage.
Inspired by people on the move
Population movement is one of the defining phenomena of our time. As mentioned in the report of the United Nations High Commissioner for Refugees (UNHCR), conflict, persecution, generalized violence, and violations of human rights have led a large number of people to leave their home, seeking for a more secure living situation. For the past years, the geographical position of the Mediterranean basin, including our country, has attracted and continues to attract a large number of influxes.
According to the International Organization of Migration’s (IOM) Flow Monitoring, there were 144,166 arrivals to Europe in 2018 with about 50,215 refugees and migrants entering Greece. These trends look set to continue in 2019 as a number of 48, 507 migrants have already reached Greece, with the root causes of displacement and migratory movements remaining unresolved [1].
During their journey to Greece refugees and migrants become vulnerable to infectious diseases due to limited access to health care in the country of origin, exposure to infections during transit, and poor living conditions in the destination country [2]. For this reason, several countries in the WHO European Region, particularly those considered as first arrival points for refugees (e.g. Greece, Italy, and Croatia), provide health screening services for infectious disease prevention. The most frequently screened diseases among newly arrived migrants are communicable diseases, and Tuberculosis (TB) in particular [3].
Current needs in Tuberculosis diagnosis
According to the World Health Organization's (WHO) annual report, TB is one of the 10 deadliest diseases worldwide, causing around 1.3 million deaths in 2017. At the same time, 10 million people are estimated to be infected by TB in 2017 [4]. Tuberculosis is an infectious disease caused by the bacillus Mycobacterium tuberculosis. It typically affects the lungs (pulmonary TB), but can also affect other sites (extrapulmonary TB). The disease is spread when people who are sick with pulmonary TB expel bacteria into the air, for example by coughing. Several factors including malnutrition, overcrowding, and disruption of health services could lead to an increased Tuberculosis disease burden [2].
Of the estimated 10 million new cases, only 6.4 million were detected and notified in 2017, leading to a gap of 3.6 million cases. These “missed” 3.6 million people are at the root of why TB transmission continues to be at such high levels [4], [5]. The underdiagnosis of TB may be due to: limited or delayed access to appropriate diagnosis and care, lack of access to appropriate diagnostic tools due to geographic and/or financial barriers, and lack of sufficiently sensitive or specific diagnostic tests to ensure accurate identification of all cases [6], [4]. Therefore, the END TB Strategy’s goals will not be achieved without new tools to fight TB.
Incorporating cell-free Synthetic Biology
According to Zsuzsanna Jakab, WHO’s Regional Director for Europe, “Refugees and migrants enjoy the same human right to health as everyone else”. Her words together with the gaps that remain in the diagnosis of Tuberculosis, inspired us to develop ODYSSEE. ODYSSEE is an in-field test for early diagnosis of TB, designed to be applied in reception and identification centers in Greece as well as worldwide.
Aiming to revolutionize commercial TB diagnostics, we had to think “out of the box”. To do so, we decided to incorporate cell-free Synthetic Biology into our design. This approach provides simpler and faster engineering solutions with unprecedented freedom of design in an open environment than cell system [7].
Biological Sample & Biomarker
The most frequently used biological samples in TB diagnostics are sputum and blood. Because of the nature of our test’s application and according to our interactions with stakeholders during our Integrated Human Practices, we decided to use a non-invasive and easily accessible sample; urine. Once Mycobacterium tuberculosis dies in a patient’s lung, it releases small DNA fragments (cell-free DNA - cfDNA) which cross the cardiovascular system and kidneys to end up in urine [8]. Our selected biomarker is the IS6110 gene, which is located in the genome of Mycobacterium tuberculosis (MTB) and encodes for a putative transposase [9], [10]. After we worked on those main pillars of our design, we had to build on our detection method.
Detection Method
ODYSSEE is an in-field diagnostic test. The “in- field” property makes it a priority for us to ensure that no special equipment and personnel would be necessary for its implementation. For this reason, the first step is an isothermal amplification method called Recombinase Polymerase Amplification (RPA), used to amplify our target gene. Using the RPA method, we achieve the incorporation of two universal sequences, adjusted to each primer as an overhang. This leads to the creation of an orthogonally engineered DNA molecule which can now enable the production of a robust signal through the following steps.
One of these two universal sequences operates as a trigger for a toehold switch. The trigger sequence binds to the molecular switch activating the expression of a downstream reporter gene through a cell-free in vitro transcription/translation system. The selection of the appropriate reporter gene was intertwined with the conditions in Low Resource Settings, like refugee camps. We chose β–lactamase as the reporter gene. It encodes for an enzyme that hydrolyzes the chromogenic substrate nitrocefin which then turns from yellow to red. This colorimetric method enables naked-eye detection of the result [11]. By combining all of the reactions described above, we developed “2tubes Philosophy”; a prototype for safe and simple in-field implementation of our test.
A universal platform
Despite TB being the most commonly screened disease among newly arrived refugees, Hepatitis B Virus (HBV) is high on the list as well [3]. HBV is a potentially life-threatening liver infection caused by the Hepatitis B Virus (HBV). It is a major global health problem and in highly endemic areas is spread from mother to child at birth, or through horizontal transmission [12]. DNA fragments derived from HBV have been reported to appear in patients’ urine. In order to prove the universality of our detection method, we tested our system in a small DNA fragment of a sequence that encodes for a DNA polymerase of the virus [13]. By changing the set of primers and making them specific for the HBV sequence, we managed to get a detection result for HBV. Therefore, we successfully proved that our system is able to detect several diseases, by only adjusting the primer set depending on the pathogenic agent targeted.
References
1. United Nations High Commissioner of Refugees. (2019). Desperate Journeys Refugees and migrants arriving in Europe and at Europe's borders
2. Kimbrough, W., Saliba, V., Dahab, M., Haskew, C., & Checchi, F. (2012). The burden of tuberculosis in crisis-affected populations: A systematic review. The Lancet Infectious Diseases, 12(12), 950–965.
3. World Health Organization. (2018). Report on the health of refugees and migrants in the WHO European Region
4. World Health Organization. (2018). Global Tuberculosis Report 2018
5. WHO (2015), Global Plan Stop to End TB. The Paradigm Shift. August 2016
6. World Health Organization. Diagnostics Technology Landscape 5th Edition, May 2017
7. Lu, Y. (2017). Cell-free synthetic biology: Engineering in an open world. Synthetic and Systems Biotechnology, 2(1), 23–27.
8. Fernández-Carballo, B. L., Broger, T., Wyss, R., Banaei, N., & Denkinger, C. M. (2018). Toward the development of a circulating free DNA-Based in vitro diagnostic test for infectious diseases: A review of evidence for tuberculosis. Journal of Clinical Microbiology, 57(4), 1–9.
9. D Thierry, A Brisson-Noël, V Vincent-Lévy-Frébault, S Nguyen, J L Guesdon, B Gicquel, Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis (1990), Journal of Clinical Microbiology
10. Thabet, S., & Souissi, N. (2016). Transposition mechanism, molecular characterization and evolution of IS6110, the specific evolutionary marker of Mycobacterium tuberculosis complex. Molecular Biology Reports, 44(1), 25–34.
11. Boehle, K. E., Carrell, C. S., Caraway, J., & Henry, C. S. (2018). Paper-Based Enzyme Competition Assay for Detecting Falsified β-Lactam Antibiotics [Research-article]. ACS Sensors, 3(7), 1299–1307.
12. World Health Organization, Hepatitis B, [Online] Available from: https://www.who.int/news-room/fact-sheets/detail/hepatitis-b [Accessed 1st October 2019]
13. Jain, S., Su, Y. H., Su, Y. P., McCloud, S., Xue, R., Lee, T. J., … Hu, C. T. (2018). Characterization of the hepatitis B virus DNA detected in urine of chronic hepatitis B patients. BMC Gastroenterology, 18(1), 19–21.
