Team:UofUppsala/Human Practices

Integrated Human Practices

Index

• Overview
• Prototype Kit Design
• Tuberculosis
• References

Which experts did we contact and what have we learned from them? Continue reading this page to figure that out!

Overview

---

Not only are cellular biosensors capable of detecting a wide range of substances, but as any other detection method, they are also highly specific (1). Through evolution, many receptors have evolved to be specific for their respective targets. This feature is utilized in our biosensor. In our project we have engineered a modular ″receptor″; a nanobody, and included several different aspects in our design on how to make the system more robust and reliable. This includes the amplification system which provides high sensitivity and rapid signalling response (1). We have a finished cellular design, but in order to create a product that could exist on the market, we have evaluated our design in terms of a commercial kit.

Throughout this project we have sought advice from experts and integrated what we have learned from them to improve our cellular biosensor. Besides these interviews, we have researched but also opened up for discussion with the public and other iGEM teams, to learn how our project affects society and how it affects us. The acquired knowledge has been integrated into the project in what is called integrated human practices.

Our integrated human practices have focused on how our device could be applied for detection necessities in the world. As one of the applications, we have chosen to see how our cellular biosensor would apply as a diagnostic tool for tuberculosis. By using modelling, we have designed a nanobody specific for the tuberculosis causing bacteria Mycobacterium tuberculosis. Below you will see the points such as Life Cycle Assessment, prototyping and case-studies for applications of our design.

Prototype Kit Design

---

Taking the step from laboratory to reality is not straightforward or predictable. Even the best experimental results have to be adapted, interfaced and applied in the way that is useful for use in real-life situations. There is no substitute for direct trial and error, but there are a few methods that can facilitate the transition painlessly. That was the topic of our event From Paper to Reality.

There, we learned about prototyping from Charlie Carpene, from EIT Health. We heard about design thinking and about the importance of understanding the context where our system is going to operate in. Our system is targeted to be used as a biosensor and we wanted to explore the possibility of developing a low cost point-of-care diagnostic system that could be operated by the users themselves. We started thinking about the places where our biosensor would be transported to and stored, what knowledge that is demanded from the user, the local health system and medical waste disposal system. From these limitations, we drew several engineering needs and proposals on how to address the problems. With these limitations in mind, we brainstormed, analyzed and explored many different concepts on paper until we found a series of designs that solved most of the known problems. We also took into consideration the design suggestions that Charlie Carpene had and integrated them into our kit. Read more about what we integrated and the design details in Engineering success! The designs are well-designed enough to be 3D printed and tested. The tests must be done in a situation as close as possible to reality, and the results must be assessed to improve the design in a later round of prototyping.

However, having an effective prototype is not enough. Any product has an environmental impact during its lifetime, either in the source of its materials, in the manufacturing process or during its disposal. To study the impact of a particular product, a different methodology is used. Life Cycle Assessment (LCA) is a powerful tool for understanding the real impact of a product beyond the laboratory. We received a marvellous lecture from Kristian Jelse from Greendesk on the basics of how to perform such an analysis.

An LCA has several phases. In the first one, the goal of the report is stated. What product or process is being studied, how far back or forward in the supply chain and general assumptions. In the second one, inventory analysis, information is compiled according to the stated requirements. This information can be obtained from public databases, previous LCA reports or internal figures of the company. This tends to be the most time-consuming step. The information is collected as inputs or outputs, and the outputs are calculated in a standard measurement of their environmental impact. For example, all greenhouse gas emissions are accounted as equivalent CO₂ emissions so that processes can be compared purely on their impact.

One difficulty of LCA is that the existing methods cover only a few potential impacts that can be analyzed objectively. It is difficult to establish standard measurements for the impacts of several outputs, such as plastic, on the health of a society or the biodiversity of an ecosystem. These methods have limitations and should be used taking that into consideration.

Inspired of the LCA, we integrated parts of it in our kit design by minimizing the dimensions to optimize the amount of material that goes into the kit. In the design, we also took into consideration to use as few components as possible to reduce the number of steps in the manufacturing and in this way minimize environmental impact. Read more in Engineering success.

Tuberculosis

---

The main infectious killer in the world is tuberculosis (TB) which, despite being preventable and curable, 1.5 million people die from every year (2). Due to TB's high impact on global health, an important target is improved diagnostics where a cost-effective, easily accessible, and improved diagnostic method would help relieve this burden.

To learn more about TB, its incidence and diagnosis, check out our pamphlet! There you will find basic information about what the disease implies, the most severely affected countries and typical methods of diagnosis. A table comparing the diagnostic methods used today and NANOFLEX can be found on the second page.

Download

Interviews

As tuberculosis (TB) causes a large number of deaths every year and is a major global health issue, we wanted to understand its effects and the needs of a diagnostic test in a highly impacted country. Thus, we chose to focus on Brazil, as TB is a significant health problem there, and met with Nuno Rufino de Sousa, a laboratory technician at Karolinska Institutet in Stockholm. He has worked and seen the issues regarding TB in Brazil and has worked on an air sampler for detection of airborne M. tuberculosis. We also met with Marlucia Garrido, a state coordinator of the Tuberculosis Control Program in Amazonas, who gave us further insight of the situation such as that Amazonas state has the highest incident rate among the states of Brazil with 76.1 cases per 100 000 inhabitants in 2019.

We received many great suggestions on how to improve our diagnostic test and much of the advice was implemented. We also discussed our idea with Leif Kirsebom, a professor at the Department of Cell and Molecular Biology at Uppsala University and a leading researcher with expertise in TB. Lastly, we interviewed Lars Hellman, a professor at the same department, who could answer our questions regarding interferon-gamma (IFN-γ). Read about these greatly rewarding interviews below!

What are the current diagnostic tests for diagnosing TB?

Nuno Rufino de Sousa

There are a few different diagnostic tests which can be carried out to determine active TB in patients, which are all relatively universal with little variation between areas around the world. The first step is usually by means of assessing one's symptoms by a doctor. Most hospitals also perform X-ray of the lungs to detect granulomas. Although it is quick, reliable, cheap and does not require sputum this means of detection is mostly only capable of detecting active TB. Other methods include molecular assays and cell cultures.

Symptoms of TB can range from prolonged coughing, fever, chest pain, and coughing up sputum and/or blood. All these symptoms act as reliable indicators.

Examination by X-ray is incredibly effective in determining the presence of granulomas, but is unfortunately not able to tell if they are due to TB. This means of detection is, mostly only capable of detecting active TB, whereas latent TB, which does not generate granulomas to the same degree, would be harder to detect, although smaller granulomas as well as scar tissue could be detected. X-ray does, however, have the advantage of being the only detection method which does not require the collection of sputum.

Two other detection methods are molecular assays or cell culture combined with microscopy. The molecular assays are mostly PCR based.

Regardless of which detection methods one chooses, prophylactic treatment will always be started before confirmation is gained.

Read more

Marlucia Garrido

For latent TB, what is most commonly done is the intradermal application of purified protein derivatives (PPD) - with no need for sample collection - in which the response of the immune system and results come out in 72 hours. Another test, where blood is collected, is Interferon-gamma Release Assays (IGRA), although it is yet to be implemented in the Amazonas.

For suspected cases of active TB, sputum is collected in most cases. With this sample, conventional smear microscopy is performed statewide. The rapid molecular test for TB (TRM-TB, in Portuguese) for TB is also performed, but only in Manaus (the capital), on the Brazil-Colombia-Peru border at the Tabatinga laboratory and in two other municipalities, Tefé and São Gabriel da Cachoeira¹. In addition to smear microscopy and TRM, we also have culture in 10 of the 62 municipalities but the sensitivity test is only done at the Central Laboratory of Public Health (LACEN) in Manaus.

¹ The state of Amazonas, has 62 municipalities, an area of approximately 1.5 million km², equivalent to three times Swedens area, and around 4 million inhabitants.

As part of the routine, a person who has been coughing for more than two weeks or who has an x-ray with some alteration suggestive of tuberculosis has to undergo sputum examination. Upon initial orientation of sputum collection, the patient collects the sample and takes it to the nearest health facility and the facility redirects the sample to the reference laboratory. There is always a laboratory with available tests where the sample can be sent to. For the city of Manaus, TRM is performed for diagnosis, whereas for cases in the inland, sputum smear microscopy is performed instead. Cell culture tests are performed in places where possible.

Read more

What are the issues with those diagnostic tests in Brazil and in areas that are highly affected?

Nuno Rufino de Sousa

Molecular assays require sputum, which only a low percentage of individuals with active TB are capable of producing. Cell culture is a method which clearly indicates the organism at hand, but it is time consuming, sensitive to contamination, and requires more work than other methods. Although it is very specific it can, with wrong conditions, take up to six months to grow and generate results. It is also very prone to contamination and generally poses more required work than other methods.

Marlucia Garrido

• If a sputum smear microscopy is performed, there are other infectious agents that may be non-tuberculous mycobacteria, fungi, and can indicate symptoms similar to that of TB and lead to incorrect treatment.
• The fastest method we currently have is the rapid molecular test (TRM) which takes 2 hours to get the results from the moment the samples are finally up for analysis. This test cannot be performed at home.
• With sputum sampling it is necessary for the professional to provide guidance for a good sample collection. Samples with poor quality lead to poor test results. But this is subject to correction by making the correct orientation of the sample collection.
• For latent TB, blood collection for IGRA itself can only be performed in the laboratory and it is not yet available in the national Public Health System (Sistema Unificado de Saúde - SUS).

In your opinion, what is more important to diagnose; active or latent TB?

Nuno Rufino de Sousa

Diagnosing active and latent TB are somewhat equally important, but their respective importance varies depending on the aim at hand. In instances where one aims to reduce the amount of infected individuals, active TB should be targeted due to the fact that it currently is unsure to what degree latent TB can spread the disease. Even though roughly one quarter of the world's population currently carries the bacteria latently, only 10% of those affected with TB will eventually actively express it. However, if an affordable and effective home kit was to enter the market, it would be preferable if it could detect both active and latent TB.

To counteract the spread of active TB, prophylactic treatment is especially important to be emphasized, but the effects of contact tracing should not be overseen. This is notably important due to how some people with active TB are more prone to spread the disease, and some even becoming “super spreaders”; infecting an abnormally high amount of people.

Read more

Marlucia Garrido

The active form. Latent does not transmit to other people. In the active form, bacteria are multiplying and disseminating in the environment. The latent is also necessary, secondarily, so that the disease does not develop.

The need for a novel test to detect active TB is more urgent. However, if a novel test were to detect both active and latent TB simulatenously, more cases could be found in time to start prophylactic treatment before the infection becomes active.

Considering our test as a home test or as a point-of-care for diagnosing TB, is there a need for it in Brazil?

Nuno Rufino de Sousa

Most of the transmission today can be traced back to specific hotspots such as schools or prisons where people cluster, which in combination with poor ventilation help the spread. The system you are developing would possibly serve as a good triage in these areas, as to not only rely on visible symptoms.

Something as simple as better ventilation can minimize the spread up to 70%, as in the case for any aerosol transmissible disease, but only so much can be done; testing for contagious diseases is also rarely, if ever, done in said areas. If people feel sick they might be directed for TB testing, but that is rather an exception to the rule as the symptoms of the early stage are similar to a flu. However, if you have been coughing for a longer period of time you would be directed for TB testing.

Read more

Marlucia Garrido

A test done at home if resembling a quick-test would be a dream come true. As of right now the only procedure that can be achieved at home by non-professionals is collection of samples; samples which require to be sent for further steps at a laboratory facility, resulting in a guaranteed 24-72-hour waiting period. And if the smear microscopy is negative, you will still have to wait for the culture results, which requires an approximate waiting period of 60 days.

Is the local healthcare system already managing the step of home-testing for TB in Brazil?

Marlucia Garrido

There aren’t any rapid tests for TB in the state of Amazonas (nor in Brazil), such as for HIV or viral hepatitis, the result of which is out in a few minutes. The quickest one is the TRM, which still takes hours to give results or days depending on the place of origin of the sample.

Nuno Rufino de Sousa

There is an ongoing global UN operation set for this very purpose. Their goal is to eradicate TB completely by 2030, as well as to reduce it to 30% by 2020. This, however, is not currently deemed to be feasible, and much more work will need to be done. They also take samples and X-rays, especially in hotspots. Other than that, people are advised to not cough openly and other common hygiene practices.

What are the difficulties regarding diagnostics and treatment of TB?

Marlucia Garrido

The treatment here is very difficult due to the long treatment time (minimum 6 months), the difficulty for health experts to monitor patients and the fact that the patient often lives a long way from the health unit. Furthermore, patients are most likely included in the group of very vulnerable populations including, immigrants, indigenous people, people on the street, as well as people with no fixed jobs and without food.

The medication causes adverse effects and when there is no food, they stop taking the medicine correctly and can abandon the treatment, leading to a very high dropout rate (15% in the Manaus, capital of Amazonas). Along with the demotivation of some professionals, it ends up becoming a neglected disease, a disease that has never left history.

Compared to PCR that lets you diagnose antibiotic resistant strains, how relevant is our idea?

Marlucia Garrido

If you manage to develop something that is more sensitive and specific, it would already help to proceed with the treatment, cure the person, and break the chain of transmission. The resistance can be identified later, but at least the treatment can already be started with the certainty that it is TB - saving time and money. There are many advantages to your idea.

Would our detection method be used in Brazil?

Nuno Rufino de Sousa

As it is an immunoassay it is harder to trust than DNA-assays due to the fact that immunoassays can generate false positives, whereas DNA-assays do not. In regards to the general public this would pose a problem, since they, of course would prefer the more reliable option. The method could hence possibly meet some resistance.

Marlucia Garrido

If validated by health surveillance, yes. People crave novelty, even more if it is something that would benefit their health. The idea would be very welcomed.

What is the cost of diagnosis for the average person? Is it available everywhere, or are there unattended regions (Amazon and other isolated places)?

Marlucia Garrido

The diagnostic tests for TB are covered by the national Public Health System (Sistema Único de Saúde - SUS), resulting in no costs to the patients. However, considering that many people live in remote areas, patients may need to spend their money on transportation, food and possibly housing, in order to get tested.

The headquarters of the municipalities have access to smear microscopy, but the tests do not reach their rural areas. Some remote places alongside the river are visited by health boats to have the samples collected and stored until they are delivered to the municipal headquarters. Even so, not all people are able to receive those visits since the boat only goes as far as you can reach by boat. Our geography is very challenging, with Amazonas demographic density of approximately 2 inhabitants/km² (compared to Sweden’s 20 inhabitants/km²). It would be revolutionary to have something that can reach these remote populations.

Read more

Although TB tests themselves in many areas might be virtually, or even completely free, many are unable to afford the cost associated with the travel or time. This further reinforces our notion of the importance of an easy and affordable home-test. You can read more about how we worked on reducing the cost of the kit in the Engineering success.

Nuno Rufino de Sousa

Some foundations, such as the Bill Gates Foundation, contribute with money to e.g. cover the cost of testing. Costs are also covered by the government. The tests are usually free if they have been ordained by doctors. They also test bigger populations and test those who are not sick for research projects.

What are your comments on our biomarker (HSP 16.3) and the technical viability of our test?

Nuno Rufino de Sousa

The target at hand, HSP 16.3, is a common one. It tends to be more expressed in latent TB, in the necrotic core of the granuloma due to stress, but is also expressed in active TB.

If you want to go with protein detection, it is actually quite hard because you need access to the protein. Taking that sample is going to be difficult in the case of latent TB, since the bacteria are located in granulomas. Something that can be looked at is the combinations of two things; both cytokine production and bacterial protein. HSP is for sure a good one, and the metabolic pathway of immune cells such as Glut1 is upregulated due to change from respiration to glycolysis or expression of IFN-γ, a golden standard for detection.

Other viable targets are antibodies specific to HSP, mycolic acid, all of which would potentially work for a home test.

Regarding the storage conditions, there is no necessary concern about nanobodies given the fact that they are exceedingly stable, as long as they are held separately to avoid cross-reactions. Long time storage might be a problem if you store at higher temperatures, but it shouldn’t be a problem at room temperature (20-25ºC).

Read more

As HSP is present in both active and latent TB we might have the advantage of detecting both forms using only one simple method. This would mean, as we previously envisioned, that we might not only have possibility of detecting active TB, but also the latent form resulting in that the disease can be treated before it becomes harmful.

Leif Kirsebom

You should not only focus on HSP as a target. There are other possible biomarkers such as tuberculin, HSP 65, RPOB (RNA polymerase), or 16S RNA. You could also look more into IFN-γ. A suggestion is to contact Lars Hellman, a professor at the Department of Cell and Molecular Biology at Uppsala University, as he knows a lot about this protein.

As the interviews suggested that we should look into additional biomarkers we started to consider a multiplexing diagnostic tool and IFN-γ, a cytokine produced by the immune system, became of interest. We therefore searched for advice from Lars Hellman to find out more about this protein. You can read more about NANOFLEX as a multiplexed biosensor in Implementation.

How viable is IFN-γ as a biomarker?

Lars Hellman

Although it in theory might be a viable option, a lot is required for its production, and it might be difficult to proceed with it as an option in vitro. This is due to the fact that the expression of IFN-γ is a step of a longer row of reactions. Diagnostic tests for IFN-γ in itself are very viable, but to have one that is mycobacteria-specific might be more difficult. Another option would be a low-weight molecule which passes through the kidneys. If you were to do that you might be able to detect it in urine!

IFN-γ is directly produced by Natural Killer (NK) cells and T helper cells which in turn is dependent on dendritic cells, so to, for example, perform clinical tests on blood to measure the change in IFN-γ concentration, one must also include all the components necessary down-stream of the chain leading up to it, which can be difficult to achieve.

IL-12, a T-helper-1 cell inducer is also important for mycobacteria responses. It, in itself, acts as a step before IFN-γ, by inducing the expression of IFN-γ. Using IFN-γ does involve the risk of presenting both false negatives as well as false positives, since both bacteria and viruses can induce this inflammatory cytokine. Some viruses can also give varying responses, some high and some low responses. It is also dependent on the individual tested. That IFN-γ gives response to so many different diseases does mean that if one were to bring forward an IFN-γ assay it could be very valuable for diagnosing several different diseases, but also have the problem of low selectivity.

Read more

What kind of sample would be most suitable to take in order to target the HSP protein?

Nuno Rufino de Sousa

To access the protein, a sputum test is probably the best and the best way of doing that is a bronchial alveolar lavage, but it is not comfortable. In this procedure, saline is put into the lungs of the patient which triggers the production of sputum.

As sputum sampling is a difficult procedure, we are considering an alternative design suited for a point-of-care diagnostic test. Read more in Implementation.

How would we in an efficient way design nanobodies or other small receptors?

Lars Hellman

We have identified a C terminal region of a lamprey protein that spontaneously forms multimers, up to 15-mers, that could be used for very efficient display of libraries of nanobodies, single chain antibodies and vaccines. Single chain antibodies can be used as good diagnostic tools as they can be produced in bacteria. The variable regions of an antibody (VH and LH) can be produced from one gene by connecting them with a flexible Ser-Ser-Gly-region that gives a functional binding region from a single transcript.

Learning that we could utilize the affinity of antibodies and make them into single chain antibodies was of great importance in the aspect that they are small enough to be expressed in bacteria. Furthermore, the repertoire of receptors for our biosensor would be expanded, not only in the case of tuberculosis detection but also for other potential applications.

Based on this information we created a guideline on how to create new nanobodies. Read more in Implementation.

References

---