Team:Rio UFRJ Brazil/Human Practices
Human Practices
The Start
Inspired by the problem caused by arboviruses, we thought to optimize the diagnostic test to be widely implemented in our country's health system and help in the early detection of these pathogens, supporting adequate medical treatment to patients. However, to get there, we need to understand the arbovirus diagnostic scenario and where to start. We evaluated the main diagnostic models through research in the literature and observation of the main methods performed by clinical analysis laboratories and chose the most used in Brazil.
Understanding the challenges of arbovirus diagnosis
During our initial research, we tried to understand which are the diagnostic tests used in public hospitals of our country. We contacted health professionals that helped us understand the diagnostics of Dengue, Zika, and Chikungunya in public hospitals.
Dr. Otilia Helena Lupi Santos, from Institute Oswaldo Cruz (IOC). During the interview, she also told us that patients search for medical care after the first week of ongoing symptoms. Then we talked about the diagnostic tests when she said that there is a 2-week detection window for the diagnosis of arboviruses. She and that in the first week molecular tests by PCR are used due to the high viral load in the patient's blood. However, it is an expensive and time-consuming method and requires accredited laboratories to perform it. The PCR tests became less specific after the second week since the viral load reduces due to the synthesis of antibodies, making identification difficult through PCR but viable by serological tests. Still, the serological tests are not specific and a cross-reaction with other flaviviruses can exist (See more) which increases the rate of misdiagnosis associated with these diseases. We asked whether differentiation between the four different Dengue serotypes was important and she answered that in the medical point of view it is not, as the treatment is the same for all serotypes.
Also, Dr. Marco Antonio Lopes de Carvalho Netto, explained that the primary screening for these diseases is carried out in the first care clinics and that it would be great to develop a portable device that could be used in these clinics. In addition, he advised us to think about the results interface, proposing that the result should be as simple as possible, ensuring that any healthcare professional could interpret the results without having in-depth technical knowledge of the procedure. He also highlighted the difference in demand for diagnostic tests in the private and public sectors. The public sector is looking after point-of-care diagnostics, which do not need a highly equipped lab. The private sector is looking for cheap and high sensitivity tests, avoiding molecular tests, such as PCR, since they are too expensive.
Juliana Bernardo Madeira, Ph.D. in Biological Chemistry, working in the Hospital Naval Marcílio Dias, gave us insights into how the clinical analysis sector of a federal hospital works. We discover that some hospitals do not have their clinical analysis laboratory, recurring to partnerships with networks of laboratories that perform more complex diagnoses. However, those who have, are automated since they perform between 400 to 500 tests per day. Also, she told us that they use immunochromatographic point of care tests to detect dengue in the emergency room since its last 20 minutes to show the results.
Dr. Otilia Helena Lupi Santos, from Institute Oswaldo Cruz (IOC). During the interview, she also told us that patients search for medical care after the first week of ongoing symptoms. Then we talked about the diagnostic tests when she said that there is a 2-week detection window for the diagnosis of arboviruses. She and that in the first week molecular tests by PCR are used due to the high viral load in the patient's blood. However, it is an expensive and time-consuming method and requires accredited laboratories to perform it. The PCR tests became less specific after the second week since the viral load reduces due to the synthesis of antibodies, making identification difficult through PCR but viable by serological tests. Still, the serological tests are not specific and a cross-reaction with other flaviviruses can exist (See more) which increases the rate of misdiagnosis associated with these diseases. We asked whether differentiation between the four different Dengue serotypes was important and she answered that in the medical point of view it is not, as the treatment is the same for all serotypes.
Also, Dr. Marco Antonio Lopes de Carvalho Netto, explained that the primary screening for these diseases is carried out in the first care clinics and that it would be great to develop a portable device that could be used in these clinics. In addition, he advised us to think about the results interface, proposing that the result should be as simple as possible, ensuring that any healthcare professional could interpret the results without having in-depth technical knowledge of the procedure. He also highlighted the difference in demand for diagnostic tests in the private and public sectors. The public sector is looking after point-of-care diagnostics, which do not need a highly equipped lab. The private sector is looking for cheap and high sensitivity tests, avoiding molecular tests, such as PCR, since they are too expensive.
Juliana Bernardo Madeira, Ph.D. in Biological Chemistry, working in the Hospital Naval Marcílio Dias, gave us insights into how the clinical analysis sector of a federal hospital works. We discover that some hospitals do not have their clinical analysis laboratory, recurring to partnerships with networks of laboratories that perform more complex diagnoses. However, those who have, are automated since they perform between 400 to 500 tests per day. Also, she told us that they use immunochromatographic point of care tests to detect dengue in the emergency room since its last 20 minutes to show the results.
Furthermore, based on what these professionals explained to us, we decided to do an interview via the internet to reach more medical doctors. We developed a google formulary focused on the diagnoses problem with the aim to understand the reality of arbovirus diagnoses in the public health system in our country. These forms were aimed at health workers, especially doctors, as they are the professionals who normally request and interpret these tests. With this, we were able to identify other problems associated with conventional methodologies, in addition to cross-reaction. We were also able to trace a pattern of stakeholder preference between one test and another.
Improving our project throught Integrated Human practices
With the data previously collected from the stakeholders, we optimized our project to solve the problems faced by the medical community (price, speed, and accuracy). So when designing Ammit , we took into account the following recommendations to achieve our purpose:
Professor Marcius Almeida from the Protein Advanced Biochemistry lab in UFRJ helped to establish which protein linkers we would use to separate each epitope found in our antigen, Ammit. We analyzed three commonly used for joining epitopes: GGG, AAY, and GPGPG. He recommended using a more rigid linker containing proline since it separates the protein domains better and contributes to protein structure stability. He also suggested verifying if the epitope structures were associated with disulfide bonds in their native proteins once this interaction helps stabilize their conformation, being difficult to accomplish the same effect in a chimeric protein. Additionally, he cleared our doubts about which selection marker we should use in the expression plasmid. He told us that ampicillin degrades with time in the culture medium, which might decrease the selection effect throughout the fermentation, being kanamycin preferred to bypass this problem. So, we decided to use pET28(a) (See more).
We also talked with the doctor in immunology, Adriana Lima Vallochi, from the Oswaldo Cruz Institute, about the difference in epitopes from B and T cells. She explained that both are important for generating the immune response, but the focus for vaccine development and diagnostics has to be different. For diagnosis, the chosen epitope has to be derived from B cells since it should be recognized by abundant clones of the antibodies. She also told us that the most commonly used epitopes are from the envelope protein and the NS1 protein and that we should search for those epitopes. Furthermore, she told us that IgG antibodies are more specific because they interact stronger with antigens, being better for diagnosis than IgM antibodies.
We also needed a way to couple our chimeric protein to a detection methodology that is different from the usual bioanalytical techniques, such as PCR and immunochromatography, and can be implemented in a point of care device. Consequently, we reached Dr. Andrea Salgado, who enlightened us about the types of biosensors and forms of signal transduction. She explained to us that our protein could be used to make an immunosensor that detects the patient's antibodies since it is made from epitopes. She also suggested that we use an electrochemical approach as a signal transducer once this methodology is used in portable devices like a glucometer. Based in this discussion, we chose to couple our protein to an electrochemical biosensor produced by us.
In our journey to develop the electrochemical biosensor, we met Dr. Fernando Henrique Cincotto, who suggested to use screen-printed carbon electrodes in our device because of its low cost and easy production. He helped us in the assembly and optimization of these electrodes using gold nanoparticles. These nanoparticles serve as the basis for the fixation of our antigen protein, contributing to increasing the robustness and sensibility of our biosensor. Also, he helped us with the tests we carried out for proof of concept with our prototype electrode, being an essential collaborator for characterization and future analyses.
We also talked with the doctor in immunology, Adriana Lima Vallochi, from the Oswaldo Cruz Institute, about the difference in epitopes from B and T cells. She explained that both are important for generating the immune response, but the focus for vaccine development and diagnostics has to be different. For diagnosis, the chosen epitope has to be derived from B cells since it should be recognized by abundant clones of the antibodies. She also told us that the most commonly used epitopes are from the envelope protein and the NS1 protein and that we should search for those epitopes. Furthermore, she told us that IgG antibodies are more specific because they interact stronger with antigens, being better for diagnosis than IgM antibodies.
We also needed a way to couple our chimeric protein to a detection methodology that is different from the usual bioanalytical techniques, such as PCR and immunochromatography, and can be implemented in a point of care device. Consequently, we reached Dr. Andrea Salgado, who enlightened us about the types of biosensors and forms of signal transduction. She explained to us that our protein could be used to make an immunosensor that detects the patient's antibodies since it is made from epitopes. She also suggested that we use an electrochemical approach as a signal transducer once this methodology is used in portable devices like a glucometer. Based in this discussion, we chose to couple our protein to an electrochemical biosensor produced by us.
In our journey to develop the electrochemical biosensor, we met Dr. Fernando Henrique Cincotto, who suggested to use screen-printed carbon electrodes in our device because of its low cost and easy production. He helped us in the assembly and optimization of these electrodes using gold nanoparticles. These nanoparticles serve as the basis for the fixation of our antigen protein, contributing to increasing the robustness and sensibility of our biosensor. Also, he helped us with the tests we carried out for proof of concept with our prototype electrode, being an essential collaborator for characterization and future analyses.
Validating our project design
We also participated in events focused on entrepreneurship in the biotechnology area as Biolatim America, Rise, and the 3FIB (Biotechnology Fair ) in Rio de Janeiro, In these events, we presented our project in the form of a Pitch, as a way to make public our project between stakeholders (MGI, Biolatim America, rise). During these events were able to stand out, which helped us to get in touch with business experts, who approved our idea, and government representatives, who financed the development of our project. We also participated in an intensive online course named Munich Global Impact Sprint (MGI) for six months, offered by the 3 Top tier universities of Munich, the Technical University of Munich, LMU, and HMU. In this event, we learned about entrepreneurship in the biotechnological area, business expertise, impact modules, and we had access to experts in the medical technology area, which helped us to start the creation of a startup.
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