Human Practices

(Informal)

Meeting with Dr. Annapurna Vyakarnam (professor of Microbial Immunology at KCL and St John's Research Institute in Bangalore, India) (conducted by Daniela)

Dr. Anna advised us to work on regions highly affected by TB in which we have contacts (the DR looking the most promising here). She recommended we speak to experts in the regions of our case studies and gather their insights on the implementation of our test. (Daniela had a meeting with a Dominican doctor as a follow-up to this.) She validated our concerns of contamination with blood samples—pushing us further away from case studies like prisons, rehabs and homeless shelters, as well as countries that lack a functional public healthcare system, like Venezuela. She spoke about her own experience treating impoverished communities with TB and mentioned a lot is down to government involvement, again discouraging us from pursuing homeless shelters, rehabs and prisons. She also had limited insight into the societal stigmatisation of TB in India, which we had researched previously and questioned her about in our meeting.

Meeting with Dr. Tayris Rodriguez (Dominican pneumologist) (conducted by Daniela)

Dr. Rodriguez was able to provide us with a deeper insight into the TB situation, specifically in the DR, discussing both cultural and societal issues intersected with the disease in the country. In the Dominican Republic, there is a big stigma surrounding TB because it is associated with HIV, which causes people not to seek diagnosis or treatment. TB is a common issue due to overcrowding. Haitian migrants are a disproportionately affected group. Electricity isn't a problem (there are not many power outages compared to other locations) here; if we wanted to implement our test it and blood testing also wouldn't be a problem if we were to implement our test in healthcare facilities (due to the sanitation that would be available there). There are many public and healthcare systems and the government also works with private enterprises to make privately administered free clinics that the public can also use. Dr Rodriguez did her residency in Santo Domingo (the most affected region) and works at a practice in Punta Cana (both coastal, crowded, poorer areas with slums—developing, majority poorer population, Punta Cana is less crowded, but there is huge socioeconomic segregation). People in the DR generally have a lot of kids in a crowded environment in poorer areas. The mother passes the disease to her children and doesn't seek care due to stigma. In her work, she found professionals in the field are not "professional"; people working chest x-rays aren't properly trained to interpret them, leading to misdiagnosis. TB is also common in children in the DR.

Meeting with Asmae Ait Abdallah (previous IGEM Team Lead and cofounder of TherAlign) (conducted by Ethan)

We spoke to Asmae, asking for advice on the progression of Human Practices following feedback we received from the iGEM mini jamboree as a previous IGEM winner. She told us to focus our project story on Scarlett's mum, who unknowingly had TB whilst pregnant (we can still use Daniela's account, but she believed Scarlett's mother's story would be more powerful). We need to get a testimonial from Scarlett's mum for this project, where we can include more detail about not knowing how she caught TB and things she would have done differently if she had known. Asmae also urged us to rethink the implementation strategy of our project. She informed us it would be difficult dispensing in hospitals as there are many national and hospital policies we need to apply to and this will not be accessible to people without access to healthcare. We can either sell our test directly to consumers or to hospitals. In distributing directly to consumers, we can use NGO-funded cars to drive to vulnerable locations to perform the tests. These cars will be equipped with generators for electricity and experts to perform the test correctly. We can also sell our test in grocers and pharmacies, but this could lead to user-related problems without expert assistance to carry out the test safely. In distributing through hospitals, she suggested we could employ a similar method to that used for colorectal cancer with postage. This has the same issue of people having to do the test themselves and also postage limitations in the DR. We could use the consumer and hospital methods simultaneously, but as it stands, distributing our test via the NGO-funded vans is the most ideal implementation plan for the DR.

Meeting with Ethan's grandma (recovered TB patient) (conducted by Ethan)

This stakeholder would like to remain anonymous but does not mind being referred to as Ethan's grandma, she provided us with an account of her unique experience of TB, which we used to educate about the diversity of TB cases. She is of Goan descent but was born and raised in Nairobi, Kenya leaving when she was 33 for England. She was diagnosed with TB in 1988, at the age of 50 (living in London). She was not previously aware of TB. She had extra‑pulmonary, non‑infectious, active lymph‑node TB. Her first symptom was a small swelling under her armpit, which grew. The symptoms that prompted her to visit the doctor were night sweats, loss of energy and weight. She was diagnosed when the doctor came to her house and performed some tests that she cannot remember in particular, but she thinks they involved taking a swab and then later the doctor called her with a diagnosis. The doctor would occasionally come to her house to check on her, as she was too sick to leave and they would weigh her as she lost a lot of weight. She didn't face challenges in accessing and receiving healthcare. When she was better, she would take a cab to the hospital to receive care. It is unclear if she contracted it here or in Nairobi, as she speculates an older relative on her mother's side also had it when she was a child, though undiagnosed; she was bed‑bound for years, and it could've been latent from then. It could've also likely been from a friend she had a close contact with. Despite Lourdes not facing much social stigma for her TB, this friend felt the need to conceal her diagnosis despite being infectious when in contact with her. Lourdes' variant was not contagious, so she experienced limited housing and employment discrimination and the doctors did chest X‑rays to check her children. She would fear needles in the case of taking blood samples due to a previous negative experience, but thinks a blood test is better than no test. However, from her experience, she doesn't believe a test that requires electricity is accessible enough. She also believed the application may be better in hospital settings.

Some additional information she shared with us about her experience was the hurdles she faced in her TB journey were: she was bed‑bound for 6 months, during this time, she was dependent on the care of her daughter. She had to take 16 tablets a day for treatment at one point. When she got a little bit better, she could get out of bed and go downstairs, but it would then take her upwards of an hour to return upstairs. She felt very weak at this time and would perspire so much that she would have to change her clothes in the morning.

Meeting with Daniela Gago (ex‑volunteer at Dominican hospital: Centro Pediatrico Oscar de la Renta) (conducted by Ethan)

Daniela's experience witnessing healthcare discrimination of Haitians firsthand was vital in informing the direction Human Practices would focus on when working specifically in the DR. Centro Pediatrico is a children's hospital that was constructed privately but managed by the Dominican Republic's public healthcare system. Daniela's work mainly consisted of shadowing doctors, supporting the children's emotional wellbeing so treatment could be administered and translating Spanish to French for Haitian immigrants. The majority of the patients she observed had respiratory diseases due to the crowded, low‑income conditions in which they lived. These patients lived in poverty or extreme poverty and most of them were Haitian immigrants. Many of these people did not have access to running water or electricity. It was a challenge communicating with them as some of them only spoke Creole and not the French used as the lingua franca. This caused a lot of frustration with the medical team due to a lack of understanding between patients and doctors. This led to problems with patient adherence to medical advice because doctors would prescribe treatments or book follow‑ups and patients would not understand and either not return or return later in a worse condition. Many of the Haitian mothers worked full‑time as housemaids and because of this, their children were often left home alone, leading to many accidents and children getting progressively sick because their parents couldn't supervise them. Due to the political situation in Haiti, many of the migrants lacked proper papers and identification. The main takeaways from this testimonial were, the public healthcare system in the Dominican Republic is mostly used by Haitian immigrants (who are the most vulnerable). The system is not equipped to deal with them, especially linguistically. We can do work to improve this by making resources in both Spanish and Creole, but more work has to be done at the government level to systematically protect this group of people.

Formal

These interviews were conducted in accordance with specific questions. Question lists for both types of formal stakeholder interviews, along with annotations on why they were asked, are available below. These interviews were instrumental in guiding the education, research and future application of our project, using real people whose daily lives have been impacted by TB to best fight TB in the real world.

Meeting with Dr. S. Lozewicz (Consultant doctor at North Middlesex Hospital with experience working with TB) (conducted by Angela)

Dr. S. Lozewicz highlighted the problems with current TB tests from their own practical experience; we used this testimony to inform how the lab and research teams could optimise our project. We were told that current TB tests are not sensitive or reproducible enough and IGRA and TST can be affected by immune status independent of the presence of latent TB. Neither IGRA nor TST revert to normal after eradication of latent TB— a potential gap in the market we can fill. TST is impractical due to it requiring multiple visits (our test is an improvement on this as it only requires one). Dr. Lozewicz uses IGRA most frequently for diagnosing latent TB and a positive culture for diagnosing active TB. There are false negatives in current tests due to immunosuppression. Additionally, patients frequently do not accept/complete the required, prolonged treatment. Dr. Lozewicz also brought to our attention that patients with inflammatory diseases planning to receive biologics are amongst the highest priority for latent TB eradication (a group we had previously not considered). In practice, social stigma of TB diagnosis exists among friends and family, especially in certain cultures. Having a test that requires the patient to travel to a healthcare centre is not accessible enough for some groups, e.g., the homeless. The main concerns shared were: sensitivity, specificity and reproducibility. We must prove the effectiveness of our test in large numbers of patients to prove our usefulness. Properly administered, blood tests can be hygienic and safe, but are resource‑intensive. Portable tests are most suited to mass screening.

Meeting with Dr. Magdelyne Ceballos (Pulmonologist working in the Dominican Republic) (conducted by Daniela)

Dr. Magdelyne Ceballos works partly in all three healthcare systems in the Dominican Republic (public, semi‑private/self‑managed and private). In their experience, TB is more common in public healthcare systems than private ones. They have been working with TB for the past 10 years, focusing more on the disease since 2020. The biggest issue they identified with current latent TB diagnostics was cost‑effectiveness. A price of $40‑100 USD isn't accessible to many Dominican families. There is also a lack of knowledge among healthcare workers surrounding latent TB; tests are not ordered and treatment and prevention of active TB are prioritised. In the DR, policy says that patients showing characteristic symptoms of TB when admitted to clinics must be screened for TB. So in pulmonary clinics, tests are more frequently ordered. Most patients are seemingly diagnosed in clinics before developing an advanced condition. We have a valid target audience with people from lower economic backgrounds, as they make up the majority of people impacted by TB and drug‑resistant TB. The main reason for this is overcrowding and limited access to healthcare services. Many of these patients don't adhere strictly to follow‑ups. These patients are treated in the public system, but it has its problems; waiting for an appointment can take up to three months. These patients also typically develop severe cases and have higher mortality rates.

Current tests are not sensitive enough (69‑83% sensitivity). There are false positives with purified protein derivatives and Quantiferon (IGRA) gives false negatives (up to 20%). The Dominican healthcare system has adopted molecular detection by PCR as a diagnostic method which is very expensive but can be provided to patients without additional cost. The government lacks incentives to diagnose latent TB. Electricity is not a limit in Dominican hospitals, as many have power plants to provide energy in the event of an outage. It is rare for power outages to last more than a day when they do happen. Use of blood is not ideal, but not necessarily a limitation, especially with a finger prick and a trained professional. Our test must be stable in the tropical climate of the DR. Shelf life must be known with specific expiration dates of reagents and time limits for interpretation. They said our test is likely to receive support from the Dominican healthcare system and lateral flow is best for mass screening.

The rate of TB in Latin America has been increasing since 2015, with higher mortality rates. At least 40‑60 new TB cases are identified annually. Santo Domingo, Santiago, San Cristóbal and Puerto Plata are most affected due to dense populations. Immunosuppressed patients with HIV and lupus, the incarcerated, military personnel, doctors and people over 65 are also very at risk. Our test would be best applied in high burden countries (like the DR) where tests are frequently performed. Many patients feel embarrassed, ashamed and worried about a TB diagnosis. This is such a prevalent issue that doctors avoid empirical diagnosis. Patients with TB are not viewed in the same light by society. Prevention of TB needs to be invested in, in the DR. Our test can help because it leads to diagnosis and treatment before the disease can progress to its active form. Drug‑resistant TB is also a common issue in the DR. Bacilloscopy and GeneXpert are both used to detect antibiotic resistance to rifampin. In 90% of cases, antibiotic resistance to rifampicin is also correlated with resistance to isoniazid. In the future, we should find applications of our test that could address issues such as drug resistance, possibly diagnosing specific strains of TB.

Meeting with Lauren Collins (former TB patient) (conducted by Ethan)

Ms Collins was a key stakeholder in our project, providing us with the story and motivation to do this project. She unknowingly developed TB whilst pregnant. If she had been aware of her condition prior, she believes things would have been very different and that she could've retained more control over both the physical and mental challenges of her journey with TB.

Upon reflection, Ms Collins believed she first contracted the disease when sharing drinks at a friend's gathering in 2009. This friend's dad was later diagnosed with TB, urging Ms Collins to get tested for the disease. Despite having a negative reaction to the TST, she believes she contracted the disease from this time and remained latent until many years later. This is likely due to the prevalence of false negatives when using TST to diagnose recently contracted latent TB. During this time in 2009, she was also pregnant and gave birth to her healthy son without any TB‑related complications.

Then, in 2014, after suffering a bad fall resulting in a collapsed lung, she believes this compromised state led to the progression of her TB from latent to active. For three weeks after her fall, she was unaware of her collapsed lung and experienced both symptoms related to her injured lung and TB. Her first symptom was visible weight loss, followed by coughing— this was originally dismissed as being due to smoking. She began having terrible night sweats, a phenomenon she had never experienced before. She felt very sick and breathless whilst walking her children to school and had slower mental processing. This prompted her to visit the hospital, where she was first diagnosed with a collapsed lung. She received treatment to reinflate her lung and in a follow‑up appointment during a chest X‑ray, she was diagnosed with severe active TB.

TB was not a danger she was aware of prior to her diagnosis. Her condition was explained to her by both a helpful nurse and personal research, leaving her shocked and devastated. At this time in 2014, she was also pregnant with her daughter. She felt she could not relate to the transmission stories the doctors shared with her of people coughing, spitting on the floor and catching TB from foreign countries— leaving her with more questions than she began with. This diagnosis and disease had a detrimental impact on both her physical and mental health. She was very concerned about her health and the health of her baby. She faced hurtful discrimination from both her friends and family. Her own mother had the perception that TB was a dirty, deadly disease from the 40s and 50s and she felt a lot of judgment. Her friends defaulted to their prejudices instead of the science and refused to visit her and bring their children around her, even when she was receiving treatment and was non‑infectious. This left her feeling isolated in a vulnerable time. For the first two weeks, she even had to remain physically isolated from her friends and family, which took a toll on her.

Her family also had to be tested and only her son came back positive for latent TB, linking with her theory that she had latent TB since 2009 when she was pregnant with him. Even though her own mother did not have TB, she was convinced she did due to her symptoms of COPD, putting a further strain on their relationship.

Ms Collins had to take 9 tablets a day for 9 months whilst pregnant, not knowing the consequences on her unborn baby. Her son had to take a liquid form of the medicine for 6 months. The school had to be notified and he had to miss 2 weeks of school and then be administered his medication during the school day. Ms Collins feels she missed out as a volunteer at the school at the time because the teachers who knew about her situation became more distant with her and would not select her for after‑school activities.

During her treatment, she had to return to the clinic every month with her son, which was logistically inconvenient, interrupting his schooling and just being generally stressful during her pregnancy. She completed her medication 3 weeks before giving birth and was confirmed clear of TB with a chest X‑ray. She felt immense relief but was warned of the risk of contracting the disease again.

Even after her recovery, TB continued to cause problems in her life. Her daughter was born healthy but a little small, so she had to be taken to the neonatal unit for two weeks. At this time, the doctors took the father to the side to ask if Ms Collins used drugs in her pregnancy, as their daughter was showing signs of withdrawal. This was obviously extremely offensive to Ms Collins, whom they had not even consulted initially and shows a clear lack of respect for a new mother. Ms Collins had to explain the medication she took related to TB during her pregnancy and things were resolved.

Now, many years recovered, she is still reminded of her experience with TB from time to time. There's ever‑present anxiety of catching it again whenever she gets a cold or feels anything that could be a potential symptom. She is also concerned that her son will be more vulnerable to the disease due to having it as a young child. Additionally, even after being recovered for more than ten years, whenever she visits the doctors, TB is always brought up.

Ms Collins' story highlights the lack of education surrounding TB, even in a high‑income country like England. Many of the people around her responded inappropriately to her condition, leaving her feeling alone at a time when both her physical and mental health were already jeopardised. Even medical professionals treating her were not sufficiently trained about TB when teaching her about transmission and dealing with TB‑related complications in her pregnancy, causing her unnecessary distress. Ms Collins said she wished she had a test like ours so she could have discovered her TB whilst it was still latent. She would have received treatment to stop it from becoming active. This could have prevented the awful physical symptoms and lessened the discrimination she dealt with, overall giving her more control and autonomy over her health. Due to the non‑contagious aspect of latent TB, she would not have been forced to reveal her condition to everyone around her, retaining her privacy and shielding herself from discrimination. She praised us for the current model of our test using blood for field diagnostics and being more accessible. She expressed a need for a test like ours back in her time. Treatment for latent TB is often less intense and draining than for active with the dual luxury of not having to deal with symptoms. She could've just taken a few antibiotics like her son for six months instead of the nine tablets for nine months.

Ms Collins is the mother of one of our principal team members; her story exemplifies the lack of resources available to people with TB and how, even in the modern day, the disease can be physically and socially daunting. A test like ours is needed to give patients like her the opportunity to retain more control over their health— a right everyone deserves.

Meeting with Hugo Fernandes (current TB patient) (conducted by Ethan)

As a nursing student at KCL, Hugo's story offers the perspective of a TB patient with extensive experience in the healthcare industry. Initially diagnosed through routine screening as part of his course, it was thought he was latent due to his lack of symptoms, but a bronchoscopy revealed lung damage and a culture from a smear showed the TB bacteria was growing inside him. The bacteria were proliferating at a rate that was too slow to be contagious but led to his true diagnosis of non‑infectious active TB. Due to the complex nature of his condition, diagnosis was a difficult and long‑winded process. He began his journey through occupational health (in his course), but had many meetings which were difficult to attend logistically, especially when he was not in London and so was ultimately transferred to the NHS. He had to have the skin test, a blood test and an X‑ray. There were long waiting times to get appointments and receive results, which took up a lot of his time. He felt he couldn't have a job at this time due to the burden of his TB, having to adhere to a strict regimen of many antibiotics every day.

It is theorised that Hugo originally caught TB from a friend who had it in school. They are still awaiting the results of the analysis of their samples to see if they are the same strain. Due to a lack of resources and time, only five people were able to be tested. The school prioritised the people he sat next to and Hugo was not included. If he had been diagnosed back then, he could have dealt with it appropriately and finished his treatment, so it would not be interrupting his life in the way it currently is today. He said this highlights the need for a test like ours to enable mass screening in the event of an outbreak. We must prioritise making our test suitable for mass screening. As someone training to work in healthcare, he was also able to offer his perspectives regarding the implementation of our test. He said the use of blood in our test is safe as long as we adhere to a sanitation protocol; he suggested including an alcohol wipe. Additionally, he said the use of electricity in our test reduces its accessibility and we should prioritise application in the field to support the most vulnerable people.

This section details some of the problems we faced with our project, brought to our attention by the numerous stakeholders we employed, and how we utilised people's accounts to overcome them. Throughout our journey, we wanted to consult people who had firsthand experience with TB and tried to amplify the voices of people whose daily lives had been impacted by the disease, giving them a platform in synthetic biology. We integrated their feedback throughout our project into research and final project design to ensure our plan and implementation would be efficient and effective as possible.

Case study selection

When deciding on case study selection we incorporated the stakeholder accounts of Daniela Gago (who had worked in a respiratory hospital in the DR), Dr Annapurna (who is a professor of Microbial Immunology at KCL and St John's Research Institute in Bangalore, India where she works directly with TB), Dr Tayris Rodriguez (a Dominican pneumologist) and Dr Magdelyne Ceballos (a Dominican pulmonologist).

The Dominican Republic was initially suggested as a case study for TB by Daniela due to her experience working there. Some of the pros of it were: our personal connection to the DR (with Daniela being a native Spanish speaker and retaining her doctor contacts— who were willing to contribute as stakeholders), being a high‑burden country the DR has many legislations trying to tackle TB and we can appease them with our project— getting support unavailable in other countries and unlike other countries we researched with high‑TB burdens, the DR has robust public and private healthcare industries which would allow us to dispense our test with the proper electricity and sanitation which was a limiting factor of our application at the time.

Dr Annapurna supported these sentiments, reiterating that we needed a personal connection to our case studies. She also discouraged us from pursuing countries where the TB situation was inherently political and we would receive decreased government support, like in Venezuela. As well as discouraging us from working in overly insecure locations like homeless shelters due to the risk of transmission of blood‑borne diseases, with a blood test. She encouraged us to speak to experts from the nations of our case studies.

Dr Tayris Rodriguez demonstrated a need for a test like ours in the DR in her testimony. She clarified that electricity and sanitisation would not limit us in the DR due to only infrequent and short power outages and the robust healthcare system. She highlighted specific areas in the DR to target: Santo Domingo and Punta Cana (her place of work) and certain communities (lower‑class urban families and Haitian migrants). She revealed problems with current tests in the DR, for example, the misuse of chest X‑rays leading to misdiagnosis in the DR due to the limited training of healthcare workers. This is something we can directly improve with our easy‑read test. She also revealed separate social issues intersected with TB diagnosis like the stigmatisation of the disease with HIV, which we thought we would be able to address in the education of our project.

Dr Magdelyne Ceballos further reiterated much of what was said by Dr Tayris Rodriguez and highlighted DR‑specific issues that our test could help, saying that electricity will not limit us in the DR (most hospitals in the DR are equipped with generators and it is extremely rare for a power outage to last longer than a day). He said in the DR, there is a lack of focused incentives on latent TB, which we can directly target. Current tests in the DR are inaccessible to the average family (ranging in price from $40–$100 USD)— our test will be cheaper.

We ultimately went with the DR due to the consensus of stakeholders and research showing a gap in the market of TB diagnosis, which we could fill, as well as problems that our test could either directly address or problems we could adapt our project to address.

Blood

Blood was a limiting factor of our test, but it was the only sample we could use, as the latent TB‑specific fold increase in RNA was primarily observed in the blood. However, blood is not an ideal sample for a latent TB test as many of the locations that have high burdens of the disease are insecure and lack the proper sanitation necessary to avoid transmission of blood‑borne diseases. Additionally, many countries with high burdens of TB also have high burdens of HIV (having HIV makes you more likely to develop active TB due to the immunosuppression). Dr Annapurna (professor of immunology working with TB in India), the general public surveys and Lourdes Rodrigues (a recovered TB patient) all brought this to our attention and agreed it was something we must address.

At first, we tried to address this via our implementation plan, dispensing our test only in hospital settings with trained individuals, which would assure safety and was an idea supported by Lourdes Rodrigues and Dr Tayris Rodriguez. However, other stakeholders, Asmae Ait Abdallah (previous IGEM Team Lead and cofounder of TherAlign), determined that a test only distributed in hospitals is not accessible enough to our target audience.

To address it we decided to scale down the RNA fold increase to be proportional to a smaller volume— this way the sample tested could be a simple finger prick (less invasive). It was also decided that we would dispense our test with sanitation protocol to reduce the risk of contamination and promote hygiene and safety. These ideas were brought about and supported by: Hugo Fernandes (a current TB patient), Dr Magdelyne Ceballos and Mrs Collins (a former TB patient).

Advantages over current methods

Dr S. Lozewicz (consultant doctor at North Middlesex Hospital with experience working with TB) spoke to us about the limitations of current TB tests that we need to address with our project: neither IGRA nor TST revert to normal after eradication of latent TB potential, BCG cross‑reacts with TST to give false positives and TST is impractical due to it requiring multiple visits.

How we designed our test to be an improvement: it does not cross‑react with BCG as it detects an RNA fold increase and is unaffected by the antigens introduced by the BCG, unlike the TST (so it won't give false positives with the vaccine like TST). Theoretically, our test should return to negative when someone has recovered from latent TB, as RNA fold increase of biomarkers should return to normal upon recovery— advantageous compared to both TST and IGRA. Our test also only requires one visit, results can be shared via smartphone and if this technology is not available to users they can simply wait for less than an hour for their results.

Discrimination

Dr Tayris Rodriguez (a Dominican pneumologist) and Daniela (who had worked in a respiratory hospital in the DR) both detailed their experiences with discrimination and social issues intersected with TB. In the DR, there is a major stigma equating TB and HIV, which prevents many people from seeking diagnosis and treatment for TB due to fear of social exclusion. Additionally, Haitian migrants face a unique position of discrimination in healthcare in the DR— being denied entry to the public system despite being the backbone of Dominican society and even in healthcare, they face linguistic discrimination, which worsens their outcomes. We targeted Haitian migrants and tried to promote education to fight the misconception that TB and HIV are equitable. We made an educational poster distinguishing TB vs HIV to fight the stigma and we also made a leaflet on the DR's guidelines for protection against TB to promote education— we made translations of the Spanish leaflet into Haitian Creole to be inclusive to Haitians.

Readout

In Dr Tayris Rodriguez's testimonial, she mentioned one of the main issues she faces with diagnosing TB (in the DR specifically) was the interpretation of current tests. In the DR, it is common to use pulmonary X‑rays to test for TB. These require some basic training. However, in the underfunded public healthcare system in the DR, undertrained healthcare workers are frequently unaware of how to interpret these chest X‑rays correctly, leading to misdiagnosis of TB. In her experience, she's witnessed this firsthand when a TB patient was given a false negative result due to user‑error by their healthcare provider. This is a particularly pressing issue since the patient would go on to believe they are TB‑free and during this time their condition would only worsen without intervention. We addressed this problem directly in our test by prioritising an easy readout method. We made the readout of our test a simple colour change in the form of a lateral flow test, which would give a clear diagnosis and not be easily misconstrued. It even indicates if the test is invalid.

Education

The findings from the quiz for the general public indicated awareness of TB but limited knowledge about the disease (as stated above). To tackle this, we made educational leaflets about TB that directly targeted gaps in knowledge revealed by the quiz. For example, the majority of quiz respondents could not distinguish between active and latent TB, so we provided information on the differences between the two in our leaflet. Additionally, we carried out an educational event at a sixth form college where we taught our audience about TB and diagnostics to raise awareness and promote education.

(Definite Limitations)

(Small sample size)

There are limited studies on biomarkers specifically for latent TB. In the study we based our project on, the biomarkers for latent TB were limited to 16 people. Small samples could potentially limit the reproducibility of our test in the greater population. This highlights the importance of TB education and awareness, as this research is valuable and people should be encouraged to pursue it and participate. Sample sizes available were also not the most diverse, mainly dealing with populations just from a single country e.g. Ethiopia. We used a study with an exclusively Chinese sample to guide the exosome isolation portion of our project. This limits our ability to compete for the inclusivity special prize due to not having diverse research. There was some diversity in the papers though, regarding age, gender, smoking and drinking habits.

(Potential Limitations)

(Lack of specificity)

We are currently unsure if our test is specific to latent TB due to a similar RNA fold increase seen in other diseases. miRNA fold increases are typically not only specific to one condition, so this limits the usefulness of our test as it could give false positives due to an individual's infection with another disease. To counteract this, we aim to market our test as a multi diagnostic tool - detecting a combination of miRNAs upregulated in LTB. This way, it can still be used for its intended purpose and we increase the accuracy to compete with other tests.

(Storage)

We want our test to have a long shelf life (at least 6 months) - similar time to a pregnancy test and lateral flow tests in the market, so we need to look into the expiration dates of our reagents. The literature paper using Cyclic Chain Displacement Reaction to amplify and detect miRNAs on a LFT included a test of storage life of 3 months and showed no significant difference in results between the two timestamps; however, unless we test this in the lab, we cannot be certain our test would be the same. Due to our aim being to do the test in one session and use the sample there and then, we wouldn't need to store samples.

(Threshold)

Our test detects miRNAs that are upregulated; however, miRNAs are present in all humans at normal levels and we did not include a system to ensure only a certain concentration of miRNA above a threshold can be detected. This could not be done at this stage as we did not work with clinical samples to find the miRNA concentration in LTB and healthy individuals to determine the threshold and if we need to adjust; however, we did propose ideas that can narrow down the threshold and asked numerous stakeholders on thoughts of our methods and the use of a housekeeping gene to normalise data.

(Blood)

Our test will require blood samples which are more invasive than other testing methods like TST but the more reliable biomarkers for latent Tuberculosis (TB) are mainly present in blood as opposed to urine or saliva. This could lead to dangers in the application of our test in low-income areas due to transmission of blood-borne disease and infection due to poor sanitation. It is also not ideal for people with a fear of needles, which is very common. Urine tests would've been more ideal in locations like prisons, rehabs and outpatient facilities where urine samples are often taken anyway. Moving forward, we need to be mindful of the implementation of our test as TB is also linked with and impacts blood-borne diseases like HIV. We will implement a sanitisation protocol along with our test in distribution to reduce poor sanitation (stakeholders ensured us this was necessary to promote safety). We will even release educational information about blood-borne diseases like HIV, to raise awareness of the safety surrounding procedures like this. Additionally, we decreased the problems that come with a blood test by making it a pin-prick so it can still be accessible to marginalised communities without requiring transport to hospitals.

The research we based our test on needed 5ml of blood, which is more than fingerpricks. In addition, centrifuges require at least 250ul of blood for separation. We will scale it down to 50ul as we are assuming that the fold change from miRNA concentration between Healthy Controls and Latent TB patients should remain the same. Scaling it down means we will need to retest the miRNA concentration in LTB patients to reconfirm the fold difference. This information can be gathered when moving our test into clinical trials. As a centrifuge is a limitation to using small samples, we are exploring microfluidics as an alternative because it can be used with blood samples as small as 6ul.

We need to ensure we have the same volume for each person; however, doing a finger prick sample makes it difficult to measure the exact volume. We can potentially implement our test into healthcare facilities where the technology is more accessible, but our aim is field diagnostics.

(Reagent toxicity)

We don't know the long-term effects of reagent toxicity of our test. Tests can be made in the lab and then sent to target locations- we need to investigate the logistics of transportation. We need to further explore the sustainability of the reagents and the potential toxicity to the environment. We need to have a protocol of how to safely dispose of reagents during testing and try to make this as environmentally friendly as possible.

(Lateral flow test)

Lateral flow tests are accessible for mass screening. The CCDR paper we based our test method on said it can be done in 15 minutes- making it more suitable for mass screening than current tests, but it wasn't tested outside of the lab as a lateral flow test, thus it is still in the testing stage.

The lateral flow test will be encased in microalgae based poly lactic acid (PLA), which is a more sustainable option for 3D printing filament material as it is biodegradable. However, the breakdown is slow and only occurs under industrial, anaerobic conditions. We will ensure our product is disposed of in industrial composting facilities so that the PLA is biodegraded to its natural components, carbon dioxide, water, and biomass, creating nutrient-rich compost which can be used in agriculture.

(Use for discrimination)

It is hard to hide a diagnosis of latent TB. Employers, landlords and immigration services could use our test to deny opportunities for people who do not have access to the proper treatment to reverse their condition. This is already done in some countries with active TB diagnosis. Our test could further marginalise vulnerable communities, denying them the chance to improve their situation.

(Limitations we can overcome)

(Multiple visits)

Both detection methods we researched and tested (toehold switches and CCDR) should be complete in one visit and the results for our final product will take 15 minutes (for LFT exclusively without . Receiving results is dependent on you having a phone. You could potentially wait an hour to receive the results in person if you don't have access to a mobile device, but this could lead to logistical challenges. Our test can also be done in two visits, but again, this faces logistical challenges in low-income communities with follow-up. Amplification of RNAs to ease detection will also add additional steps to our test, making it longer.

(Negative policies)

Our previous research into case studies involving the homeless could have negative impacts on policies affecting them due to it repeatedly implicating informal living settlements as primary areas of TB transmission. This could potentially lead to induction of policy banning such settlements, which would be extremely discriminatory to the homeless. However, now this is no longer our primary case study this should be less of an issue.

(Electricity)

Our test had a potential need for electricity, which could have limited its accessibility. If we were to use a centrifuge for the extraction process, this would require electricity but there are battery-operated centrifuges; however, many low-income countries do not have the equipment necessary for this in public hospitals (e.g., Venezuela), so this was a limiting factor of where we can implement our test. This technology is more readily available in other high-burden countries like the DR, though. Need for electricity limits application in field diagnostics (e.g, homeless shelters, outpatient facilities etc.).

To make our test suitable for a low-resource setting, we changed our extraction process to microfluidics. Microfluidics can separate plasma, isolate the exosomes and extract the miRNAs without the need for electricity so this is no longer a limitation. Due to our aim being to do the test in one session and use the sample there and then, we wouldn't need to store samples, hence a refrigerator is not needed.