Team:Vilnius-Lithuania/Implementation

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PROPOSED IMPLEMENTATION

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Implementation

Our project is a two-level solution to amebiasis. The first essential step is prevention, which with minimum effort reduces the risk of getting an invasive, life-threatening form of amebiasis. Probiotics would protect people from ailments caused by amebiasis and prevent further disease progression. The capsule possibly should be consumed 30 minutes before food or beverages containing fat, as studies show this is the best approach for bacterial survival [1]. We also took into account our stakeholders’ insights, that we should raise awareness about the disease and wanted to justify reasons why the prevention is important. Therefore, along with the instruction for the usage of probiotics, an educational leaflet would be added. We considered the notice that some people in developing regions might have trouble reading, therefore, we included many illustrations.

Visualised amebiasis symptoms: fever, dehydration, stomach ache, blood in stool, diarrhea. Visualised additional (besides probiotics) prevention methods: wash hands after sneezing or coughing, starting work, touching face, handling raw meat, visiting the toilet, handling money, do not prepare food if ill, have clean hands and wear clean clothing when working in the kitchen, thoroughly wash fruits and vegetables, use boiled water, have a minimum of 74 degrees Celsius temperature for meat preparation.
Fig. 1. Educational information about the importance of the use of probiotics

If the prevention is unsuccessful or missed, then the diagnostic test can be applied. The designed test will be affordable for the majority of natives, will ease the diagnosis and help to confirm or reject amebiasis diagnosis, by differentiating E. histolytica caused infection from other similar symptoms presenting ailments. The test itself would be easy to use and not require any specific knowledge. Therefore it is going to be applicable to point-of-care testing in various situations. The instructions for the usage of the test are presented below. Here we also considered that some people might have trouble reading and added as many as possible illustrations.

Amebye test instructions: 1. Disinfect finger; 2. Prick the finger; 3. Take blood with a pipette; 4. Add blood to all three holes of the test; 5. Wait 20 minutes to get the result; 6. Flip the test to get the results. Interpretation of the results: the test has three circles, which colors indicate the result. If the middle circle is red, then the person is tested positive for amebiasis. If the middle circle is blue and there is one red circle, the person is tested negative for amebiasis. If all circles are blue, then the test is not functional.
Fig. 2. Instructions for using the test

Lastly, to clarify the implementation process of the diagnostic test in the real world, we have written a business plan. Our chosen business model is out-licensing. By implementing this plan, we would patent a developed diagnostic test and sell its rights to a strategic partner for further development, production, and marketing. You can find out more about our business plan in Supporting Entrepreneruship page.

End users

One of our project targets is natives in the tropical regions (Mexico, South Africa, India, Egypt, etc.) because there are the highest rates of amebiasis, and they have the highest risk to encounter E. histolytica in their closest environment. This target group could use probiotics and diagnostic tests as they are user-friendly and do not require any specific conditions for prolonged storage. After consultations with dr. Ieva Giedraitytė and Mantė Makauskaitė we have learned that the most effective way of reaching natives is through community leaders, local doctors, teachers, who have earned the community trust.

This brings us to our next target group - health care centers. Such institutions are going to benefit from the diagnostic test as it will be low cost and rapid; therefore, fast diagnostics and treatment strategies will be possible. In addition, this test is useful not only for the health care centers of endemic regions but also for all other countries where imported cases occur. We have been acquainted with this information from the integrated human practices activities, in particular, during consultations with the head of the infectious diseases diagnostic laboratory Jolanta Žiliukienė. Firstly, we have been targeting only the native people and health care centers of countries in endemic regions, but after the conversation with J. Žiliukienė, we were pleasantly surprised that this infection and the problem of its detection occurs in our own country too. So, our diagnostic test would facilitate rare but still occurring cases screening.

Furthermore, we should not forget that the people who travel around the world seeking exotic vacations sometimes get deadly diseases instead. This typically occurs in the areas where they could afford to pay for the medicine or screening, but there might be a lack of knowledge or qualified personnel to perform complicated tests. And here comes naringenin-producing probiotics, which would protect travelers during their trips, reduce undesirable stress, and curb the spread of amebiasis.

Finally, our software EFBALite + TEA is easily reachable in framework HuggingFace for AI community who seeks to fine-tune it for a similar task that includes the search for any length aptamer up to 30 nucleotides that are highly affinite to their target protein or just need better fundamental aptamer list for SELEX method.

Read more about our insights from societal perspective on our Integrated Human Practices page).

Safety and other considerations

One of our project’s products is a genetically modified probiotic which is capable of efficiently conducting naringenin synthesis. In order to control the spread of this GMO organism, we have designed a kill switch with the toxin/antitoxin VapXD system [3]. Expression of the VapD toxin is under the control of a temperature sensitive promotercspA. If probiotic bacteria escape the intestine and enters the environment, it is going to be killed by intracellular exoribonuclease VapD toxin because cspA promoter enables transcription when the temperature is lower than average of the human body. However, the promoter might be leaky. To solve this, VapX antitoxin expression is controlled by bile salt inducible promoter. In this case, if the bacteria are in the intestinal, there are enough levels of bile salts to induce antitoxin production and sustain probiotic viability. Altogether both promoters work in synchrony when producing toxin and antitoxin to enable cell survival when needed and suppress them in any other not specified location.

Furthermore, our designed test is based on the blood sample analysis, which requires blood sample acquisition from the finger. This procedure needs to be as sterile as possible to prevent any infection. Since our primary focus is point-of-care diagnostics which enables users to conduct testing that does not depend on the medical personnel, together with a test, we provide a clean needle, pastette to draw the blood, a disinfectant wipe, and a bandaid to sterilize the puncture wound. Even with these precautions, human error can always occur, and there is still a chance of infection.

There are some other considerations needed to be taken into account. Firstly, our probiotic product is considered a GMO as it has some particular changes in the genome. This creates some legislative restrictions in the EU even considering that it could be named as a food supplement. However, in tropical region countries, there might be less problems with the legalization of this product. Although the part of society is still keeping the distance from the food or medicaments containing GMOs, in the past year, hostility toward those products usage has dropped remarkably [2] opening the opportunities to successfully implement our designed probiotics into the real-word.

Moreover, we need to measure the clinical significance of our probiotics and diagnostic test. During the clinical trials, we will find out if our probiotics are efficient and safe to use and if the test is specific and sensitive enough to generate reliable results. For now, we can only assess whether probiotic bacteria produce naringenin and wether diagnostic test does not generate false-positive results.

References

1.
Tompkins, T., Mainville, I., & Arcand, Y. (2011). The impact of meals on a probiotic during transit through a model of the human upper gastrointestinal tract. Beneficial Microbes, 2(4), 295–303. To the article.
2.
Ichim, M. C. (2021). The more favorable attitude of the citizens toward GMOs supports a new regulatory framework in the European Union. GM Crops & Food, 12(1), 18-24. To the article.
3.
Bertelsen, M. B., Senissar, M., Nielsen, M. H., Bisiak, F., Cunha, M. V., Molinaro, A. L., ... & Brodersen, D. E. (2021). Structural basis for toxin inhibition in the VapXD toxin-antitoxin system. Structure, 29(2), 139-150. To the article.