Government officials
Dr. Yi-huah Jiang
Description
Dr. Yi-huah Jiang is a professor in the Institute of Strategic and International Affairs at National Chung Cheng University and also the former premier of the Republic of China. With his professional background in political science, he knows how government policies are formulated.
Purpose
When we first started our project, we had a discussion with Dr. Jiang. We wanted to know what we should do and how he would consider our project from the perspective of the government.
Input
He told us that the accuracy of a detection kit is the foremost consideration of the government. Furthermore, the cost of the detection kit is also an issue that will be taken into consideration as the government’s policy decisions are based on the taxes paid by the public. He also warned us that a dengue virus detection kit is a medical device categorized as class II, requiring premarket review to get a license before being allowed to go public. So, he suggested that we contact the Taiwan Food and Drug Administration (TFDA) to get more information about verification of the detection kit.
Adjustment
We understood that besides improving the accuracy of our product, reducing the price will directly benefit the government and the public. At the same time, we started to consider the reliability and how to implement our project in the long term.
Taiwan Food and Drug Administration (TFDA)
Description
Taiwan Food and Drug Administration (TFDA) is a government agency in charge of the quality and safety of food, drugs, medical services and cosmetics. The department of medical devices is responsible for verification, registration, and inspection of medical devices.
Purpose
As the dengue virus detection kit is classified as a Class II medical device, it can only be sold on the market after applying for a medical device license. We wanted to know what kind of verification we should achieve in order to apply to put our product on market, so we consulted with De-Syuan Chen, Ph.D. and Jin-Yu Lee, Ph.D., who work in the medical device and cosmetic group, on the standard of verification for a dengue virus detection kit.
Input
They gave us an introduction on how they verify and inspect medical devices. For a dengue virus detection kit, we should first conduct preclinical trials to show the safety, effectiveness, and quality of our product before applying for a license.
They further pointed out precautions we should pay attention to in our product:
- We had to find out what metabolites might interfere with our product, such as fucose and mannose. We had to take them into consideration and try to prevent interference.
- We should decide what kind of liquid sample we are going to use, since each has a different type of interference, so we had to consider the corresponding pretreatments.
- The virus particles in real life would be much different from the modeled ones, while the structure of the peptides might change after the conjugation to the gold nanoparticles.
Adjustment
We had a better understanding of the process of verification and license application for a detection kit. We also realized that there were plenty of problems we should tackle to apply our project to a real life situation. We should confirm how we would collect and pretreat the blood samples in order to prevent interference. Thus, we decided to consult with related units which would use a dengue virus detection kit.
Department of Health, Kaohsiung City Government
Description
Although it previously had one of the highest numbers of dengue cases in Taiwan, the government of Kaohsiung City has put extensive effort into the prevention and control of dengue, giving Kaohsiung City mature measures and technologies for dengue control.
Purpose
To know how important a role a detection kit plays in the control of dengue outbreaks, we visited the Department of Health of Kaohsiung City Government. We also consulted with them on the procedure of using detection kit use and the steps taken after a dengue fever case is reported.
Input
They introduced the concept of three levels of prevention, including the primary, secondary and tertiary prevention.
Primary prevention aims to inhibit disease before it occurs by limiting the risk of exposure of healthy individuals, for instance, by cleaning stagnant water in containers. Secondary prevention, also known as screening, aims to reduce the impact of a disease by early detection. Tertiary prevention efforts focus on people already affected by the disease and attempt to reduce the severity of the disease to prevent disability and restore functionality.
From them, we found that our project is classified as the secondary prevention. They emphasized that this stage is important in preventing the worsening and the spread of this communicable disease. Moreover, medical expenses and psychological burden can also be decreased thorough the early detection and the early treatment.
They also explained to us how dengue cases are reported and the process of dengue fever detection. There is a dengue fever reporting system in Taiwan. Every case of dengue will be reported to the system so that each related unit can take action as soon as possible. Apart from prompt medical care, actions that should be taken include vector surveillance, environmental disinfection, and elimination of mosquito breeding sources. Furthermore, they have to draw a density map of mosquitoes and investigate the TOCC (Travel history, Occupation, Contact history, and Clustering) of patients to prevent further outbreak of dengue in the community. Thus, once a patient is diagnosed with dengue infection, lots of actions need to be taken, which will involve many different units such as hospitals, Taiwan Centers for Disease Control (TCDC), the local health bureau and the local environmental protection bureau.
They were also willing to share their difficulties in dengue prevention. They told us that it is sometimes hard to convince the public about the importance of environmental disinfection and vector elimination, especially the elderly, because they lack awareness of this disease.
Adjustment
We realized that apart from medical units, our project can also bring great benefits to dengue prevention and control units. This really inspired us to help them by researching and developing a detection kit with high sensitivity and specificity so that their efforts would not be in vain!
We also learned about the process of detection and how the dengue reporting system works, which helped us to understand more about the application of our project to society.
From them, we realized that we lacked knowledge about public health and preventive medicine. We learned that a combination of primary, secondary, and tertiary prevention is important for prevention and protection of health problems. This lead us to conduct our education and engagement about dengue based on these three concepts so that we could have a deep impact on raising the awareness about dengue. Moreover, their difficulties in outreach to the elderly prompted us to conduct a series of education programs targeting an elderly community.
Experts
Dengue Prevention and Control Center, Tainan City Government
Description
Dengue Prevention and Control Center was established by the Public Health Bureau of Tainan City Government, with the aim of coordinating government agencies’ participation in investigation and control of dengue. The center is in charge in epidemic analysis, vector monitoring, and chemical control of the vectors.
Purpose
We wanted to know the perspective of a dengue prevention and control center on using a dengue virus detection kit and their demands for a detection kit, so we consulted with Miss Ling-Sai Tsai, who was section chief of the unit and her team. We also shared our project with them.
Input
They said that a certified, useful detection kit had to achieve certain standards:
- Detecting in a short time
- High sensitivity, above 80%
- Low false positive and false negative rates
- Low cost, allowing mass production
- No side effects
We consulted with them about pretreatment of the blood samples. Although there is more interference when using whole blood, they suggested we use whole blood in our product. Since there are no centrifuges in developing and remote areas, it is more convenient to use whole blood for the on-site detection. They also shared their experiences that the patients with hyperlipidemia and those taking the liver medications would interfere the detection, so they would ask whether suspected dengue patients were taking those medicines before using the detection kit.
Adjustment
We decided to use whole blood in our product. We discovered that apart from early detection, we needed to improve the quality of our product. We should improve the sensitivity and specificity, and more importantly decrease the false positives and false negatives of our product. Thus, we designed and conducted experiments to test the false positives and false negatives of our product. Moreover, we knew more on the interference of our product, which urged us to address on them.
Kaohsiung Medical University Chung-Ho Memorial Hospital
Description
As Kaohsiung City has previously encountered a dengue outbreak, Kaohsiung Medical University Chung-Ho Memorial Hospital had abundant clinical experience with dengue. The hospital also established the Tropical Medical Center, which focuses on the study and investigation of tropical diseases, including dengue fever.
Purpose
As medical institutions are the front-line workers for dengue patients, we wanted to know the medical treatments undertaken after dengue cases are reported and their perspective on using a dengue virus detection kit. Moreover, we hoped to know how important a detection kit was in medical care from their point of view. Thus, we had an interview with Jih-Jin Tsai, MD. Ph.D., who is the managing director of Tropical Medical Center and her team.
Input
Patients will be classified into groups based on their severity. Patients who are not that serious will be advised to care for themselves at home while patients who show warning signs or may develop into severe dengue will be hospitalized and receive medical care.
From them, we found out that although there is no specific medicine for dengue currently, the mortality rate of severe dengue can be decreased to below 5% with proper medical care. On the other hand, the root cause of dengue mortality rate was delayed diagnosis. That’s why dengue virus rapid detection kits are distributed to primary care institutions to ensure that dengue patients can be diagnosed at an early stage. Furthermore, to prevent spread, a rapid detection kit is crucial during an outbreak period.
They shared their experiences of using a detection kit. They said that the sensitivity of the detection kit will be affected by the serotypes of dengue viruses and whether the patients have a primary infection or secondary infection. Other flavivirus such as Japanese encephalitis and Zika virus will also affect the detection results, due to cross-reactivity.
They also shared their experience with international medical aid in the Solomon Islands. They established a dengue diagnostic system and medical treatment system by teaching them to use diagnostic technologies such as the NS1 detection kit and PT-PCR test.
Adjustment
We got the picture of the arrangements made for dengue patients in hospitals. We discovered that a dengue virus detection kit is an important and essential index in judging dengue infection, especially during a dengue outbreak. We also gained a thorough understand of the problems faced by health care workers using the current detection kit. That helped us to figure out what type of detection kit would fit their needs. They also expressed a willingness to collaborate with us by providing serum for free testing if our product was completed.
Moreover, their experiences in the Solomon Islands made us understand the difficulties and challenges in implementation of dengue medical diagnostic system in an undeveloped area. However, we still explored the great potential of Dendetx in the overseas market, especially developing countries that were at risk of dengue. We believe that our product could bring beneficial changes to those countries!
National Mosquito-Borne Diseases Control Research Center (NMDC)
Description
The National Mosquito-Borne Diseases Control Research Center (NMDC) was established by the National Health Research Institutes of Taiwan in response to the severe dengue outbreaks reported in southern Taiwan in 2014 and 2015. It plays an active role in helping epidemic-control units of both the central government and local governments by providing them with useful knowledge concerning evidence-based epidemic prevention measures.
Purpose
We hoped to determine the feasibility of our project so we consulted with Chun-Hong Chen, Ph.D. working in NMDC and his team. At the same time, since they are experts in surveillance, control, and prediction of vectors, we wished to know further information on technologies they used and how they think our project could be used in vector surveillance.
Input
They pointed out the key problem of our project is the affinity between CLEC5A and E protein. We have to prove that CLEC5A could really bind to the E protein or else our project would have no significance.
Furthermore, they think that it is quite feasible to apply our project to vector surveillance. They shared their experiences and efforts in that area. They told about the tough nut they faced, which is the difficulty of vector detection when using the NS1 detection kit. As the NS1 antigen of mosquitoes is not released from the cells, researchers have to destroy the cells before detection. This makes it impossible to carry out the detection at the scene without a professional measurement instrument. Thus, they said that our detection kit could bring huge benefits to them if our kit could overcome that problem.
Adjustment
We understood that proving the feasibility and rationality of our project is important if we want to use our detection kit in real life. This spurred us on to simulate the affinity between CLEC5A and E protein to ensure that our experimental basis is valid. Moreover, we also simulated the binding between our peptide and E protein so that our project can be proven workable. (More information on Model)
Academia
Lai-Kwan Chau, Ph.D.
Description
Lai-Kwan Chau, Ph.D. is a distinguished professor in the Department of Chemistry and Biochemistry at National Chung Cheng University. He is an expert in the field of analytical chemistry and material chemistry.
Purpose
The affinity between peptide of tandem-repeated sequences (PTRSs) and gold nanoparticles is the key question we have concerning the detection kit. We want ensure that the gold nanoparticles could flow through the absorbent pad but not accumulate in other places. To resolve those problems, we consulted with Dr. Chau.
Input
We asked for his advice about the affinity between gold nanoparticles and PTRSs and also the mobility of gold nanoparticles on fibers. He suggested that we use mercaptans (R-S-H) to modify the surface of PTRSs before conjugating them to the gold nanoparticles. He also suggested that we use hydrophobic nitrocellulose fibers so that the samples could flow through the fibers.
Adjustment
We took Dr. Chau’s suggestions and decided to use hydrophobic nitrocellulose fibers on the control line and the test line of the detection kit.
Yen-Ta Tseng, Ph.D.
Description
Yen-Ta Tseng, Ph.D. is a postdoctoral researcher in the Department of Chemistry and Biochemistry at National Chung Cheng University. His expertise is analytical chemistry.
Purpose
We wished to have a comprehensive understanding of the preparation and modifications of gold nanoparticles. Thus, we decided to consult with Dr. Tseng.
Input
We were not sure what is the proper size of gold nanoparticles to be used in our device. He told us that particles in this kind of application usually range between 13 nm to 15 nm. He suggested that we prepare the gold nanoparticles by reducing chloroauric acid with sodium citrate. He also suggested we use mercaptans with a carboxyl to form a self-assembled monolayer on the gold nanoparticles. Moreover, he reminded us to keep the fibers thin enough to make sure the sample could flow through the fibers successfully.
Adjustment
We decided to use 13 nm gold nanoparticles. Moreover, we learned how to prepare and modify the gold nanoparticles from Dr. Tseng. Under his guidance, we succeeded in obtaining the gold nanoparticles and modifying the self-assembled monolayer.
Zhi-Xian Wang, Ph.D.
Description
Zhi-Xian Wang, Ph.D., a postdoctoral researcher in the Department of Chemistry and Biochemistry at National Chung Cheng University.
Purpose
In our original proposal, we planned to use nitrocellulose fibers as the material for our test line. But we found that the bonding between nitrocellulose fibers and the peptides is a dipole-dipole interaction, which is a very weak interaction. We had to find a way to improve the interaction between the test line and the peptides.
Input
Dr. Wang shared his experience of modifying glass fibers with carboxymethyl-dextran (CM-Dextran), so carboxylic groups can be attached to the surface of the glass fibers. The glass fibers were further modified with EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide)/NHS (N-hydroxysuccinimide), which can bind to the primary amines of DNA.
Adjustment
We realized that glass fiber membranes are a sheet of glass fibers, so we should be able to employ the method mentioned by Dr. Wang. Thus, we decided to use glass fiber membranes instead of nitrocellulose fibers as the test line in our product and follow their modification procedure.
Since CM-Dextran also contains polysaccharides, they could also interact with the peptides. Therefore, we decided to replace CM-Dextran with carboxyethylsilanetriols (CES). CES also has a carboxylic group that can be modified with EDC/NHS, allowing the test line to form a covalent bond with the primary amines of peptides, which is a stronger interaction than the dipole-dipole interaction.
Problems We Met
After the modification of gold nanoparticles using mercaptans, we observed serious aggregation. All the gold nanoparticles formed a deep purple precipitate, which prevented further experiments.
Thus, we consulted with some professionals below:
- Zhi-Xian Wang, Ph.D.
Dr. Wang advised us to coat the gold nanoparticles with CM-Dextran, which would avoid the aggregation of gold nanoparticles. However, if the gold nanoparticles could not aggregate at all, they would lose the ability to play their role as an indicator.
- Dr. Sugi Guthula (Ph.D. in Chemistry and Biochemistry, expert in applications of nanoplasmonic biosensors)
Dr. Guthula suggested we use MHDA (mercaptohexadecanoic acid)/SB-SH (1-(2-sulfosulfanylethylamino)tetradecane) to modify the gold nanoparticles. MHDA has a long-chain hydrocarbon, which forms a monolayer on the gold nanoparticles, and therefore, has a lower tendency for specific absorption. Meanwhile, the EDC/NHS approach still can be applied to the carboxylic group from MHDA for further interaction with peptides. Thus, we decided to modify our gold nanoparticles with MHDA/SB-SH.
References
PŘISTOUPIL, T. I.; KRAMLOVA, M.; ŠTĚRBÍKOVÁ, J. On the mechanism of adsorption of proteins to nitrocellulose in membrane chromatography. Journal of chromatography A, 1969, 42: 367-375.
Saftics, A., Prósz, G., Türk, B. et al. In situ viscoelastic properties and chain conformations of heavily hydrated carboxymethyl dextran layers: a comparative study using OWLS and QCM-I chips coated with waveguide material. Sci Rep 8, 11840 (2018).
JOHNSSON, Bo; LÖFÅS, Stefan; LINDQUIST, Gabrielle. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance
WANG, Lin, et al. Fluorescent nanoparticles for multiplexed bacteria monitoring. Bioconjugate chemistry, 2007, 18.2: 297-301.
CHIANG, Chang-Yue, et al. Fiber optic nanogold-linked immunosorbent assay for rapid detection of procalcitonin at femtomolar concentration level. Biosensors and Bioelectronics, 2020, 151: 111871.
Public
Overview
If dengue fever reaches epidemic proportions, it would not affect only a small part of the population, as the whole of Taiwan is in the zone of potential infection. Therefore, to make sure our research can not only affect and benefit society but also be socially responsible, we need to know how much Taiwanese citizens know about this disease. Do they understand its severity? Do they agree that this disease is something we need to be concerned about? Understanding of the public’s thoughts on this issue would help in our follow-up actions.
Moreover, to ensure that our detection kits meet the needs and are suitable for use here, we wanted to understand the current status of dengue diagnostics and whether there is a need for a dengue virus detection kit. Meanwhile, since our project involves synthetic biology, we wanted to know how well the public understood it to determine how easily they would accept the application.
To get that information, we conducted two surveys at different times.
The First Survey (May)
We did our first survey in Taiwan, focusing on understanding how much the public knew about dengue and synthetic biology. To understand their thoughts well, we used both quantitative and qualitative methods. For the quantitative method, we created a questionnaire composed of closed-ended questions that were a mixture of single or multiple choice, checkbox, and matrix questions. We shared our questionnaire online, and also distributed questionnaires at Chiayi Cultural Park and Chiayi Train Station, which are the most highly trafficked areas in Chiayi City. For the qualitative method, we randomly selected some Taiwanese citizens to conduct in-depth interviews to gain a deeper understanding of their views.
We collected 248 questionnaires for the quantitative analysis. Among the population sample we collected, nearly 50% were 20~39-year-old citizens, and over 80% of people had at least a high school education.
The results showed that over 70% of people did not realize that they were living in an area susceptible to dengue fever. Also, nearly 70% of people did not know about severe dengue. Moreover, nearly 70% of people did not know what kind of treatment is required for dengue fever.
What's even more regrettable was that most people randomly interviewed think that dengue fever is not serious in Taiwan and that it can only occur in the south. This is not as the same as the information Ministry of Health and Welfare verified, because their information indicates that due to global warming the range of mosquitoes is expanding, so infections in the north are increasing. Furthermore, all of the local dengue fever infections this year happened in the northern part of Taiwan, which confirmed their misunderstanding of dengue fever.
In terms of synthetic biology, we noted that 70% of people had never even heard of it.
To summarize the results, we found out that more than half of Taiwanese citizens lack common understanding and vigilance about dengue, even those with higher education. Moreover, they have a huge misunderstanding about this disease, leading to the thought that they believe it is unimportant. It makes us realize that we have to popularize the knowledge about dengue fever so that they would pay attention to this disease. This made us hold a series of outreach and education programs about dengue fever. If we could start with education, the public would know how serious dengue fever is, which would raise their acceptance of our project, knowing it could change the situation.
Meanwhile, we hope we could raise the public’s understanding of synthetic biology so as to improve their acceptance of our project. Therefore, we also planned to popularize synthetic biology with education and publicity.
The Second Survey (September)
We conducted a second survey in order to investigate the current use of dengue detection kits and the public’s need for a detection kit. In this survey, we collaborated with Team iGEM IISER Berhampur, which was also working on dengue. We shared our questionnaire online, and spread the questionnaire to more places with the help of other iGEM teams. We collected 303 questionnaires for total.
We asked them how soon they would consult a doctor for a fever with muscle and joint pain, which are the typical symptoms of dengue, to know the estimated period when a detection kit would be used. From the survey data, we found that most people would seek medical treatment from the first day to the fifth day of the symptoms, which is the period (period of viraemia) when our detection kit has the highest sensitivity!
Moreover, we asked about what price they would find acceptable for a dengue virus detection kit. 53% would only accept a price below $7, while another 44% would be willing to pay up to $15. When choosing a dengue virus detection kit, the factor they consider most important is high sensitivity and specificity. The next most important factors are the ability to diagnose before symptoms emerge and inexpensive.
Thorough this survey, we developed a better understand of the public’s demands in a dengue virus detection kit. This directed us to develop a detection kit that can provide an accurate diagnosis early and inexpensively!
