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Rapidemic

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Description

Inspired by the current COVID-19 pandemic and the lack of accessible testing, we set out to develop of a new, point-of-care rapid diagnostic kit. Throughout the past couple of months, we studied the field of molecular diagnostics to gather information and performed experiments, which eventually led to the preliminary proof for our detection method. On this page we will discuss our project background, goals and where we are now.

Introduction

In March 2020, coronavirus disease 2019 (COVID-19) was declared a pandemic by the World Health Organization (WHO) only a few months after its discovery. It was clear that the world was not prepared. Countries tried their best to contain the spread but were quickly overwhelmed as the first wave made its way across the globe. Within a couple of months, an obscure disease had managed to weaken public health care systems, damage economies, and leave millions without a job1-4. Nonetheless, control over the disease could be regained through effective measures, such as social distancing, lockdowns, case detection and contact tracing, which resulted in a reduced number of infections5,6. However, isolation of the population is not a long-term solution due to its severe consequences for the economy as well as for the daily income of many7,8. Testing has therefore become more and more important as long as no effective vaccine or treatment is available. Nevertheless, as testing increased, laboratories were quickly overwhelmed and the reagents required to perform the PCR tests became shorter in supply. This left us with a lack of accessible tests, which formed a major challenge of global importance. This has inspired us to develop a rapid diagnostic test as a point-of-care alternative for PCR tests. In doing so, we hope to enable a rapid and effective response to future outbreaks.

Diagnostic testing is important to 'flatten the curve'. Frequent testing enables the detection and subsequent isolation of infectious people. This strategy effectively reduces the number of infections and postpones the peak in number of infected people.

Try out our interactive epidemiological model to see how important a rapid diagnostic response is for controlling an outbreak.

Importance and shortcomings of testing

Preparation is essential to respond quickly and effectively to outbreaks in the future. Infectious diseases travel faster than ever in our increasingly globalized world9. Moreover, deforestation rapidly increases the contact between humans and animals, resulting in a smoother transmission of zoonotic pathogens10,11. Finally, global warming affects the abundance and distribution of disease vectors as a result of changing environmental conditions, which allows some vectors to expand their geographical reach while others disappear12,13.

Global changes happening in our world, such as global warming, globalization and deforestation, provide more opportunities for new emerging viruses.

Attempts to effectively contain the spread of infectious diseases have failed due to the lack of rapid, accurate, and accessible diagnostic testing14,15. Currently, diagnosis is routinely performed with polymerase chain reaction-based (PCR-based) tests16,17. PCR-based tests are accurate and fast, but the requirements for laboratory equipment and expert personnel increase the overall test time to over 24 hours17. The need for laboratory capacity is a restricting factor in population testing, especially in low-resource areas where lab testing is sparse15,18. Point-of-care rapid diagnostic tests exist, but these antigen-based tests often lack sufficient sensitivity, and their clinical use for detecting COVID-19 is currently strongly discouraged by the WHO19–22. In addition, the development of antigen-based tests takes longer than that of a PCR test23, while a rapid diagnostic response at the start of the outbreak is essential for successful outbreak containment14,15. To be prepared for potential future epidemic-causing pathogens such as listed in the WHO R&D Blueprint or unknown diseases collectively referred to as “Disease X”, there is an urgent need for innovative tests that can quickly be adapted to new pathogens24. Text adapted from our preprint.

For an in-depth comparison of existing tests, please visit the Entrepreneurship page!

Rapidemic

Inspired by the current COVID-19 pandemic, we developed a new bio-molecular technology for an accurate, point-of-care rapid diagnostic test, called Rapidemic. The aim of our project was to design a test that is laboratory-independent, provides results fast, and could easily be adjusted to test for any epidemic-prone pathogen. In doing so, we integrated the WHO criteria for rapid diagnostic tests in our kit, abbreviated as ASSURED: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users25. Furthermore, generic parts of our kit could already be mass-produced and distributed globally in advance to get ahead of the next pandemic, while a target-specific part of the kit can be distributed as soon as the pathogen's genome sequence is known. Visit the Implementation page to read more about the production and distribution of the test.

The bio-molecular technology of the test consists of three well-established techniques. During the first two reactions, a specific part of the pathogen's genome is amplified and a DNA-based reporter is produced. In the third reaction, the reporter provides a clear visual readout in the form of a color change (see figure below). The combination of the three reactions to detect target DNA or RNA forms the inventiveness and the novelty of the technology.

Concept version of the Rapidemic test kit and its properties. Rapidemic's most important characteristics are: 1) provides fast and accurate results; 2) is rapidly adaptable to new targets; 3) is suitable for use in point-of-care settings. Similar to PCR tests, Rapidemic's nucleic acid amplification method provides accurate detection. The primers involved in the amplification allow fast adaptation to new targets by changing the primer sequence. The amplification occurs at constant and low temperatures, which enables testing outside a laboratory with no need for specialized equipment. The representation of the kit's hardware in the figure is still in development.

FAST AND ACCURATE

Similar to PCR, our technology is based on nucleic acid detection, as this method is more sensitive than antigen-based testing26-28. Specific nucleic acid amplification leads to the production of a short sequence of single-stranded DNA that folds into a specific structure with enzyme-like activity. This DNA-based structure acts as the reporter of the system. Unlike protein-based reporter systems, we avoid a protein translation step which allows for a short reaction time29.


RAPIDLY ADAPTABLE TO NEW TARGETS

The technology is specific to the pathogen of interest due to the use of amplification primers that target a specific sequence in the genome. Depending on the set of primers that is used, this generic yet modular system can be modified to fit a multitude of pathogens. Since the design and validation of a new primer set are more straightforward than the development of an antigen-based test, our technology allows for a rapid diagnostic response to a newly spreading disease30.


POINT-OF-CARE

To avoid the need for laboratory equipment, we combined the colorimetric readout with isothermal DNA amplification methods. In contrast to PCR, such amplification methods are performed at a low and constant temperature to avoid the need for a thermocycler. Moreover, the specific method in our technology has among other methods one of the lowest working temperatures, close to room temperature31. The point-of-care design and low cost of the technology make the test accessible to anyone, regardless of their geographical location.

The project

Our technology integrates three bio-molecular reactions and applies them as a novel method to detect the presence of a target sequence. During laboratory experiments, the three reactions were coupled in a serial manner by using the product of each preceding reaction as a component for each subsequent reaction. After several rounds of optimization, we performed a proof-of-concept experiment to show how the three reactions can successfully be connected to detect, amplify, and report the presence of a targeted DNA sequence. The experiment provided the preliminary proof that our sequence detection scheme can work as intended. Subsequently, we designed and constructed a prototype test kit to show how the technology holds the potential to be further optimized into a rapidly adaptable point-of-care diagnostic tool. Lastly, an empirical model was developed as a kinetic representation of our sequence detection kit. This model was used to assess the performance of our kit, troubleshoot remaining problems, and guide future optimization of our technique.

Prototype of the Rapidemic test kit. Three color reactions are visible through the prototype's window. From left to right: a sample reaction (blue), a negative control (transparent) and a positive control (blue).

About 8% of all men and 0.5% of all women are affected by color blindness32. Thus, it is important that the color change of the test result is colorblind-proof! But do not worry, the color change that our technology provides is visible to anyone. Fun fact: Every figure on this wiki including our logo is color-blind friendly as well. Curious how and why we did this? Take a look at the Inclusion page!

High epidemic potential: respiratory-borne RNA viruses

We had various interviews with stakeholders that helped us to shape our project. We learned that a test that can be adapted to a multitude of pathogens would be very helpful, but from an entrepreneurship point-of-view, a modular kit that has no specific application would be complicated to market. Therefore, we decided that we had to start small and establish ourselves with a specific application and gradually build up the product to its final, modular form. We settled on adjusting our method to detect respiratory-borne RNA viruses, for instance influenza viruses. This group is highly prone to mutations and its epidemic potential poses great public health risk33,34,24. The high mutation rate is something that can be addressed well by the modular nature of our kit. A rapidly adaptable point-of-care test like Rapidemic would be very well-suited for influenza as an example, as the antiviral drugs depend on the influenza strain and treatment provides the most benefit for patients within 48 hours of symptom onset35,36,37. Ultimately, if we prove that our kit can be well-implemented for a specific respiratory-borne RNA virus, we hope it increases the credibility of our modular application, build up a reputation and accelerate the approval and implementation of our kit for new diseases in future crisis situations.

Future prospects

While COVID-19 has brought inspiration for our project, it also limited us in our ability to perform experiments and left us with several paths that have remained unexplored. Throughout the time in the laboratory, we came across several issues with regard to the sensitivity and specificity of our detection system that need improvement, which you can read about on the Engineering and Results pages. We also aim to transform the technology into a one- or two-pot reaction to simplify the prototype design. Subsequently, we continue our research into the hardware design by brainstorming with specialists about possibilities for an affordable yet user-friendly test kit. Altogether, we intend to truly fulfill the potential we believe our diagnostic kit possesses as an innovative alternative for currently existing point-of-care diagnostic products. Eventually, we hope to provide users with a high-quality, easy-to-use device that can be deployed around the world to help prevent the next outbreak from becoming a pandemic.

References

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  2. Smith E. Global stocks head for worst week since the financial crisis amid fears of a possible pandemic [Internet]. CNBC. 2020 [cited 7 October 2020]. Available from: https://www.cnbc.com/2020/02/28/global-stocks-head-for-worst-week-since-financial-crisis-on-coronavirus-fears.html
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  4. Coronavirus Disease 2019 (COVID-19) [Internet]. Centers for Disease Control and Prevention. 2020 [cited 7 October 2020]. Available from: https://www.cdc.gov/coronavirus/2019-ncov/global-covid-19/task-sharing.html
  5. Khanna, R. et al. COVID-19 pandemic: Lessons learned and future directions. Indian Journal of Ophthalmology. 68(5), 703 (2020).
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  7. Nicola, M. et al. The socio-economic implications of the coronavirus pandemic (COVID-19): A review. Int. J. Surg. 78, 185–193 (2020).
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  17. Corman, V. M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance 25, 2000045 (2020).
  18. Guglielmi, G. The explosion of new coronavirus tests that could help to end the pandemic. Nature 583, 506–509 (2020).
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  28. Keipp Talbot, H. & Falsey, A. R. The diagnosis of viral respiratory disease in older adults. Clinical Infectious Diseases 50, 747–751 (2010).
  29. iGEM Team EPFL 2019. Retrieved on Oct 10, 2020 from https://2019.igem.org/Team:EPFL/Design.
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About Us

We are the 2020 team of iGEM Leiden. With an interdisciplinary team of students we aim to develop a point-of-care rapid diagnostic tool for infectious diseases!

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iGEM Team Leiden
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igem@science.leidenuniv.nl

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