Team:Thessaly/Applied Design
PRODUCT
DESIGN.
Simple, rapid, robust and affordable diagnostic solutions are the first step on a journey to alleviating and treating endemic diseases, thus enabling entire populations to be freed from the shackles of illness. Tuberculosis (TB) is one of the 10 deadliest diseases worldwide that mainly affects vulnerable populations, such as migrants and refugees. Due to the lack of innovative solutions for the early diagnosis of TB leaning on the principles of Synthetic Biology, we developed ODYSSEE. ODYSSEE is a TB diagnostic test destined to be applied in refugee camps. By developing a two-way dialogue with stakeholders including Non-Governmental Organizations (NGOs), the World Health Organization (WHO) and doctors from our nearest General Hospital, we learned about the shortcomings that they face and managed to incorporate their needs into our product’s design. ODYSSEE embodies the features of a triage test that can be used by first-contact healthcare providers to identify those who need further testing. The universal nature of our design makes our test capable to detect more than one disease, offering health workforce an innovative solution for disease screening.
Our design process
Click to each step to find out more...
Framework
Throughout history, Tuberculosis (TB) has persisted because its roots are deeply intertwined with economic and social inequalities. Measured by the numbers of people who die each year, (nearly 1,6 million people, meaning 4,400 victims every single day) TB is one of the world’s deadliest infectious diseases [1]. This curable and preventable disease known to humanity for thousands of years remains a major health problem for our community, despite the progress made in global TB control.
Tuberculosis (TB) is caused by bacteria (Mycobacterium tuberculosis) that most often affect the lungs. TB is spread from person to person through the air, when people with lung TB cough, sneeze or spit. It is enough for a person to only inhale a few of these germs to become infected [2].
The global number of new and relapse TB cases notified and the notification rate per 100,000 population have both been increasing since 2013 as shown in Figure 1. Despite these increases, progress in closing detection is slow and large gaps remain. 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 [1], [3].
Figure 1. Notifications of TB cases (new and relapse cases, all forms) (blue) compared with estimated TB incident cases (purple). 2000 – 2017, globally and for WHO regions [1]
The underdiagnosis of TB may be due to; limited or delayed access to appropriate diagnosis, failure to test for TB when people do present to health facilities and, lack of access to appropriate diagnostic tools due to geographic and/or financial barriers [1], [4]. Most of the currently available TB diagnostics are not fully adapted to resource-limited settings or specific patient needs and a lot of them are priced out of reach [4].
Among the people who have limited access to quality TB diagnosis are refugees and migrants. Tuberculosis is a leading health threat for populations affected by crises, and is the most frequently screened disease among the newly arrived migrants [5], [6]. For this reason, according to the Foundation for Innovative New Diagnostics, “to reach more patients we need novel tests that are more rugged, easier to use, and that can penetrate deeper into the health care system than current tools”.
Existing Solutions
Although the TB diagnostics pipeline appears robust, with several technologies in development, no new platform has emerged in 2018. Technologies under development are primarily molecular-based. Τhey include tests used to detect; TB for diagnosis and treatment monitoring, drug resistance among confirmed TB cases, and TB and drug resistance simultaneously, in centralized high-throughput laboratories [1].
In recent years, WHO reviewed and endorsed the use of several tests for TB diagnosis that are meant to be used at different levels of care. Along with these tests, in Table 1 there are displayed and briefly described the methods that are commonly used in health facilities. These methods mainly refer to Greek’s Health System about which we had the opportunity to discuss with several healthcare specialists.
Table 1. Existing solutions on TB diagnostics [4], [7], [8], [9], [10]
Although the introduction of these methods represents a step towards TB diagnosis, some critical limitations remain, regarding their use in the most decentralized areas, and especially refugee camps.
Many of the methods above are invasive or use biological samples that are difficult to collect, such as blood and sputum respectively. These constraints concern mainly the younger population, which corresponds to a large number of people infected with TB. Doctor Dimitris Patestos, working with Doctors of the World – Greece, specifically claimed that: “Children can find it hard to produce sputum, and when they do, it is often low in bacterial load” (Personal Communication 31 January 2019). On the other hand, despite the fact that LAM Test is based on the detection of Lypoarabinomannan (LAM) in urine samples, its exclusive use in HIV positive populations limits its application in a wider spectrum.
Expensive instrumentation, uninterrupted energy requirements, and the need for specialized personnel, confine the effective deployment of methods like XPERT/RIF and smear microscopy in rural areas that lack this kind of infrastructure. Another important aspect in the diagnosis of TB is the timely delivery of results, which is critical for earlier treatment initiation. Methods such as the tuberculin skin test, well known as the Mantoux test, and liquid cultures fail to deliver results on the same day. During our visit to the ICU General Hospital of Larissa in Thessaly, in February 2019, we talked with the pulmonologist Dr. Karayiannis for the most commonly used method in-field, Mantoux. He pointed out that this test requires a second visit to a health specialist in order for him to examine the reaction’s results that may be controversial. This qualifies as an important disadvantage for Mantoux as a TB screening tool. On top of that, Eirini Gerogli, Coordinator of the Migration Health Division at IOM (Study visit 13 May 2019), informed us that in refugee hotspots in Greece, there is a lack of tuberculin reagents so the test cannot be conducted.
Considering all these, we understood that the landscape of TB diagnostics lacks the operational characteristics that would allow the implementation of current diagnostic technologies in lower levels of the health systems. Despite the great effort for development, there are still gaps to be filled for achieving healthcare accessible to all.
Our design thoughts
During our project development, we came across with a set of Target Product Profiles (TPPs)TPPs specify the clinical purpose of the test, the goal to be met, the target population that will be tested, the level of implementation in the healthcare system, and the intended end-users. In addition, TPPs should outline the most important performance and operational characteristics [11]. Both WHO and the TB community have agreed on these TPPs as factors defining the performance and operational characteristics a test should meet, in order to respond to the needs of target populations [3], [11].
At the same time, we visited the International Organization of Migration (IOM) (13 May 2019), an organization with great experience in working with vulnerable populations, such as refugees and migrants. There, we met with Eirini Gerogli, Coordinator of the Migration Health Division. She mentioned that a simple low-cost test that can be used by first-contact providers to rule out TB, such as a triage test, could be a very useful tool in these settings. Furthermore, Dr. Apostolos Veizis from the Doctors Without Borders (personal communication 10 May 2019) specifically claimed that in Reception and Identification Centres (RICs) there is an urgent need to minimize the referral to the nearest hospital. He stated that, in order for the health system to improve its capacity in response and preparedness, a simple test that could sort out those who need further testing would be a really helpful tool.
Taking all these into account, we decided to develop a triage test, rather than a confirmatory test, adjusted to the needs and infrastructure of a refugee camp. In order to better direct our product’s development, we set up 5 important requirements that our product should meet.
Our Solution - ODYSSEE
Patients’ access to new diagnostic tools must be considered alongside with product development. In our effort to bridge the gap between science and patients and turn our idea into reality, we incorporated the feedback from all the stakeholders together with our team’s principles into our product design.
We created ODYSSEE, a simple and easy to use triage test for early diagnosis of Tuberculosis, specifically designed to be applied in refugee camps and limited resource areas. ODYSSEE is aimed at being used in RICs by first-contact providers in the community with the purpose to rule out TB and direct individuals who require further examination to a confirmatory test.
The technology behind the test
ODYSSEE, utilizes the principles of cell-free Synthetic Biology exploiting the presence of DNA fragments (cell-free DNA) of the Mycobacterium tuberculosis in patients’ urine.
Our test comprises three main steps; amplification, in vitro transcription/translation, and an enzymatic reaction that enables the visualization of the result. Diving into the molecular level, the amplification step aims to amplify the specific MTB gene IS6110 with the use of an isothermal amplification method called RPA (Recombinant Polymerase Amplification). In the amplified product, unique signal sequences are incorporated. Once this product is transcribed, these sequences enable the activation of a toehold switch. Toehold-switches are RNA devices that can be activated only in the presence of a specific trigger sequence leading to the production of a reporter protein, after the translation stage. In our effort to associate design with application, we selected as the most suitable reporter protein β-lactamase, an enzyme able to hydrolyze the chromogenic substrate Nitrocefin. This enzymatic reaction leads to a color change from yellow to red, allowing the result to be seen with a naked-eye.
These three steps carry unique operational characteristics that led us to incorporate them into our product design, always considering its implementation in Resource Limited Setting.
Furthermore, taking into consideration Apostolos Veizis’ view of screening diagnostic tools, we managed to create a universal platform for diagnosis. During our conversation, he claimed that the purpose of a screening test is to be able to detect multiple diseases at the same time. For this reason, our proposed designed test is able to detect other diseases, based on DNA, by only changing an element included in the workflow. We successfully proved that our design works also in DNA fragments of the Hepatitis B Virus (HBV) derived from patients’ urine.
Check out our Demonstration Page to learn more about our validated results.
Implementing our technology
ODYSSEE, as an end-product, incorporates all our design requirements. Viewing our product from the perspective of a potential end-user, in this case, being a healthcare provider at a refugee camp, we came up with “2tubes Philosophy”. With this philosophy, we envision transforming the complexity of our lab’s experimental steps into a product ready to be applied in-field. In this way, we are able to minimize both the need for special training and the cost. Learn more at our Entrepreneurship page.
Figure 2. The 3D design of our prototype in AutoCAD and Rhino 6
In most cases, limited resource settings lack of a proper electricity system. In order to overcome this problem and offer an opportunity to use our test across the globe, we are developing a heater. Our upcoming heater is aimed at keeping the temperature of the tubes constantly at 42oC. This allows our detection test to operate in optimal conditions. Thus, we propose a solution that ensures portability and can be applied in the locations of interest, thanks to a heating device that operates with batteries. After our visit to George Charalampous (study visit 20/9/2019), Assistant Professor in Thermofluid Processes with Energy Applications at the Department of Mechanical Engineering of the University of Thessaly, we concluded that our heater will have the following characteristics.
Characteristics
• Designed to have spots for 20 tubes.
• Contains 20 thermistors as heating elements, which are placed around the tube spots.
• Functions with either electrical outlet or batteries.
Why thermistors?
The resistance of a thermistor depends on temperature, more than in standard resistors. Thermistors are widely used as self-regulating heating elements. Through the process of Joule heating, thermistors convert electrical energy into heat.
Why placed in spiral?
In order to keep a constant temperature throughout the tube, at any point of the tube. Moreover, heat between the thermistor and the tube is transferred from the hot environment to the cold one, complying with the second law of thermodynamics. The aim of that is to equalize the temperature difference, in our case, from the thermistor to the tube.
Below you can find our detailed product guide and the instructions' scheme of our test.
As part of a patient-centered care, all patients should receive support from mediators on the field, help them undertake the test. In terms of ending discrimination and encouraging integration, we provide our product guide in different languages. To do that, we first asked from the National Public Health Organization (NPHO), which are the most commonly spoken languages amongst refugees arriving in Greece. Then, we reached out to our fellow foreign iGEMers from this year to ask for their collaboration regarding the translation of our guide.
Demonstration
Once we designed our prototype, in collaboration with 3D Hub Volos, we had it 3D printed (Figure 3). Aiming to demonstrate our product and the way it’s used, we visited the Accommodation Facility “Agia Eleni”, located in Ioannina, Greece. “Agia Eleni” is a structure specialized in the assistance of the most vulnerable among refugees and is under the “umbrella” of the Non-Governmental Organization, INTERSOS. During our visit there, we had the opportunity to conduct a simulation of our test together with the hygiene specialists and find out whether it is easy and safe to use.
Figure 3. Our 3D printed prototype
During the simulation, we pointed out that our prototype does not bear all the features of its final implementation, as it was developed during a limited time. Furthermore, respecting iGEM’s safety policies, we didn’t use any biological sample or reagent but demonstrated the steps of the test using pure water.
Once we finished our demonstration, Iraklis Telidis, the Senior Accommodation Supervisor of “Agia Eleni”, conducted the test on his own, by following the steps included in our product guide. Both he and the audience confirmed that our test is easy and safe to use to implement. Iraklis is not a healthcare provider, so in this way we proved that the personnel executing the test does not need to be specialized. Moreover, the discussion that followed the simulation, gave as feedback on how we can improve our design. The healthcare providers working with the National Public Health Organization (NPHO) pointed that the universality of our detection method is really useful for in-field diagnosis. They stated that is highly possible that healthcare services would be willing to invest in an easy to use and timesaving tool for TB screening. Moreover, Nikola Skoula, hygiene specialist of INTERSOS, underlined the issue of TB vaccination and its affect on our test’s result (false positive). She advised us to find out which strain is contained in the vaccine and maybe use it as a positive control. She gave us food for thought by commenting on our test’s detection and implementation. Finally, the social workers that were present during the implementation, stated that it was really thoughtful of us to provide a product guide translated in different languages, in terms of contributing to the communication and trust issues met at refugee camps.
Figure 4. Simulation of our test at the Accommodation Facility "Agia Eleni" for refugees at Ioannina, Greece
Choosing to work with Mycobacterium tuberculosis, a pathogenic microorganism, and its diagnosis, we should take strict safety measures. For this reason, safety has accompanied our product throughout its development regarding its usage, the end-user, the patient, and the environment, as well.
Biological Sample: Sputum, as a sample, contains alive bacteria which could potentially cause disease transmission, in case of sample misuse. Furthermore, blood sampling is an invasive method risking the patient’s disapproval for undertaking the test. In order to overcome these implications, our test uses urine as the biological sample; an easily accessible, non-invasive body fluid.
Biomarker: The cell-free DNA we decided to detect, which derives from Mycobacterium tuberculosis, is harmless and non-pathogenic.
Cell-free System: The use of a cell-free system instead of live bacteria helped us avoid any hazardous effects. Our system does not involve any potentially harmful biological parts and its sub parts are approved and classified as nonhazardous.
Biosensor Device: By using a Synthetic Biology riboregulator, the toehold switch, we manage to regulate gene expression using a unique trigger sequence. This way, there will be no expression of the reporter gene if the unique synthetic activator we have designed is absent, even if those parts are accidentally released into the environment.
Reusable elements: Aiming to take a step towards protecting the environment, many of our test’s components (Pasteur Pipette, Syringe, Heater etc.) are reusable and recyclable, after the proper sanitation.
Product Guide: Our product is supplemented with a detailed and informative product guide in order to ensure the right execution of our test and minimize the risks of human error. In our product guide we also point out the need for a biohazard bin for safe disposal of the test’s components. This way, the release of harmful reagents into the environment is prevented.
Index Card: In terms of privacy, our product guide contains an index card aiming to help healthcare providers match the number of the test they are executing with the right patient. In order to protect the patient’s personal data, we are indicating the use of a barcode instead of the patient’s name. Note: This feature doesn’t fully ensure the patient’s privacy and more measures should be taken.
Responsible Use: ODYSSEE is a triage test and not confirmatory. In such way, after its execution further examination is needed. On the other hand, potential users of our test should be right informed and educated about its use and the announce of the results, able to offer psychological support.
Impact on society
“Many of history’s greatest successes in the fight against preventable diseases have been characterized by momentous shifts in people’s belief of what is possible. What shifted and made these successes possible? It was the belief that change was not only possible, but necessary.” Words by Dr. Aaron Motsoaledi and Dr. Joanne Carter, chair and vice-chair of the Stop TB Partnership, respectively.
Regarding the impact of our project on the world, we created ODYSSEE in a way that it contributes to the goals set by the End TB Strategy, supported by the World Health Organization (WHO), and the United Nations 2030 Sustainable Development Goals. To make a step further, we developed “2tubes” Philosophy promoting simplicity into our community.
Contributing to the END TB Strategy
In 2014, the World Health Assembly unanimously approved the End TB Strategy, a 20-year strategy to “end the global TB epidemic”, with the vision of a world with “zero deaths, disease and suffering due to TB” [3]. The strategy comprises a package of interventions organized under three pillars and four principles that necessitate the collaboration between governments, communities and civil society organizations in an ethical, equitable, and human rights-based approach [15].
As indicated in Pillar 3, ending the TB epidemic will require modern and better tools for its detection. For this reason, we urgently need to endorse research and innovation in order to turn sophisticated laboratory technologies into robust point-of-care platforms. This way, TB diagnosis will become more accurate and widely available for patients and care providers [15]. ODYSSEE makes a step towards this achievement by introducing cell-free Synthetic Biology to TB diagnosis. This innovative field enabled us to develop a de-centralized, portable diagnostic platform that can be rapidly conducted in-field by minimizing the requirement of specialized equipment and personnel met in laboratories in large urban centers. This way, we introduce a new and innovative tool for the early diagnosis of TB to the healthcare workforce.
Another important issue is that some groups in high risk for TB, such as migrants, face stigma and discrimination, and may avoid seeking out care out of fear of social or legal repercussions. Reaching this kind of key populationspeople who are vulnerable, underserved or at‑risk of TB infection and illness [3] will be essential for ending TB [15]. Furthermore, among the END TB Strategy’s principles is the use of a human rights-based approach that includes respect for ethical values and pursuit of equity. Alongside OSYSSEE’s development, we aimed to involve these vulnerable populations as key stakeholders in the fight against the disease. We conducted our research with ethical standards and provided equitable access to a new tool for TB diagnosis. Finally, in terms of product development, we managed to address the underlying inequities of communities who bear a greater burden of TB infection, as they are mentioned in the END TB Strategy.
Implementing the pillars of the End TB Strategy requires intensified action from and beyond the ministries of health; action taken in close collaboration with all stakeholders including other ministries, communities, civil society, and the private sector [15]. Bearing this in mind, we propose a healthcare model supplementing our test implementation, in order to promote a multidisciplinary approach for the issue of migration and its health aspects.
Contributing to the 2030 Sustainable Development Goals
Back in 2015, at the General Assembly of the United Nations, world leaders agreed to 17 goals for a better world by 2030. These goals are known as the Sustainable Development Goals (SDGs) and focus on the development of global cooperation between governments, businesses, civil society, and all of us, in order to build a better future for everyone [16].
These are the main SDGs that ODYSSEE is contributing to. However, we must stay aware that incorporating appropriate TB responses into the efforts that are made to meet some of the other SDGs will accelerate the end of TB. We must bear in mind that we ought to be supportive, empathetic, inventive, passionate, and above all, cooperative, in order to achieve the Global Goals.
“2tubes” philosophy: describing the ability to do more with less
We often become witnesses of an excessive approach to the world of materials and products. Unnecessary sources overwhelm the market by giving the impression of high comfort and functionality. However, in iGEM Thessaly we are trying to adopt a different belief system regarding the world of industry and products. We believe that the usage of minimum materials can result in perfect functionality. The combination of solid aesthetics and pure beauty were some of our team’s values that led us to design ODYSSEE.
Our product, ODYSSEE embodies our perspective of how we see the world; simple, safe, and caring. Putting in the center of its creation the potential user’s interest, we managed to integrate the stakeholders’ feedback into our design. We placed all the information and suggestions in a common line, driven by our philosophy of minimalism, and our goal for ODYSSEE to be adapted based on society’s needs. Two tubes allow for the simplest way to execute our test, with a rack used to help on their management. Commonly used materials simplify the test’s usage at a maximum level. In such a way, the overall experience of the user when interacting with our test, will be less stressful and more engaging.
Our key goal is to achieve “more” with “less”. With ODYSSEE, we can appreciate the functionality that simplicity can offer. Let’s disaffiliate the draining and endless competition for “more” and integrate the simplicity both into our products and our lives.
Future vision
The design of a prototype is just the beginning. We should always look into the future and keep trying to overcome some implications and improve the user’s experience. Below, we are stating some of our ideas aiming to upgrade our product.
Create our own cell-lysate: In order to minimize the cost of our product, we aim to create cell-lysates for the in vitro transcription/translation system. Cell-free expression systems can be prepared using either a cell extract, or a combination of purified recombinant proteins, and be done at 37 – 42oC optimally. This method provides a 98% cost reduction compared to comparable commercial cell-free systems, approximatively 0.03$ per μL instead of 0.79$ for PURExpress that is now used in our test [17].
Add a sample pretreatment step: Estimating the M. tuberculosis cfDNA concentration in biological samples is vital for assay development, including sample preparation (sample collection volume, cfDNA isolation method, and concentration volume) [18]. Our research should be focused on the development of the right sample pretreatment method.
Incorporate our positive control: A suitable control in our test’s design would eliminate the possibilities of false positive or false negative results. This is why we chose cox3 gene deriving from the human mitochondrial DNA, for positive control. The advanced version of our test should contain this control in order to further ensure its reliability.
Detect more diseases in one-test: As we successfully proved that our method is capable of detecting both Tuberculosis and HBV, we aim to incorporate into detection of both diseases in our product.
For scaling up our test we are thinking about developing an origami paper-based diagnostic test. The purpose of this is to add all the above components into our test.
References
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3. WHO (2015), Global Plan Stop to End TB. The Paradigm Shift. August 2016
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6. World Health Organization. (2018). Report on the health of refugees and migrants in the WHO European Region
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11. WHO Report, M. (2014). for New Tuberculosis Diagnostics: Report of a Consensus Meeting. WHO Meeting Report, (April), 1–98. https://doi.org/WHO/HTM/TB/2014.18
12. Lillis, L., Siverson, J., Lee, A., Cantera, J., Parker, M., Piepenburg, O., … Boyle, D. S. (2016). Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care. Molecular and Cellular Probes, 30(2), 74–78. https://doi.org/10.1016/j.mcp.2016.01.009
13. Li, J., Macdonald, J., & Von Stetten, F. (2019). Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst, 144(1), 31–67. https://doi.org/10.1039/c8an01621f
14. New England Biolabs (NEB), PURExpress® In Vitro Protein Synthesis Kit, , [Online] Available: https://international.neb.com/products/e6800-purexpress-invitro-protein-synthesis-kit#Product%20Information [Accessed 18/10/2019]
15. WHO. (2014). Implementing The End TB Strategy. Antimicrobial Agents and Chemotherapy, 58(12), 7250–7257. https://doi.org/10.1128/aac.03728-14
16. The Global Goals for Sustainable Development, [Online] Available: https://www.globalgoals.org/ [Accessed 11/10/2019]
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18. 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.
