Team:BOKU-Vienna/Human Practices

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How Human Practices have shaped our 2019 Project:

From idea generation to the design of a Buruli Ulcer diagnostic test kit

Martina Helmlinger
for the iGEM BOKU-Vienna team 2019
Vienna, October 2019

Acknowledgements

The iGEM BOKU-Vienna team is eternally grateful for all the input and support received from many different actors. Without their help, the project would not have come nearly as far as it has, and we would like to express our gratitude for these generous provision of time, expertise and encouragement. Thank you!

 

Executive Summary

Insights and feedback from diverse stakeholder have strongly influenced the development of our project from the start. After investigating our project’s position within the international efforts of Buruli ulcer research and mitigation, we approached experts in medical sciences, health care providers, molecular biologists, ethicists, social scientists and former patients to discuss and finetune our system. The results were considered in the design of our test kit, from the level of Genetically Modified Machines (GEM) to the kit level and beyond. These considerations have guided decisions in our project work in various ways.
The most notable output was the test kit and manual, which were carefully drafted based on the exchange with experts and the lab team.
The input and dialogue have also shaped ideas for future steps beyond what was possible during this year’s competition, and the considerations shaped both the wet-lab part of our project and other decisions. The iGEM BOKU-Vienna team hence has successfully integrated the inputs from diverse experts and other stakeholders into their project.

 

Table of Contents

1. Considerations and Development
2. The three layers of our project progress
3. Safety considerations
4. Dialogues which have shaped our project
5. References

 

Part A: Project Outline and considerations

1. Considerations and Development

1.1 Introduction to the diagnosis of Buruli ulcer

Buruli Ulcer is a hard to detect disease in Africa and some parts of South America. It is caused by Mycobacterium ulcerans, and it owes its symptoms to Mycolactone. It can lead to lasting lesions and deformations (WHO, 2019). Thus, without question, giving attention to this neglected disease felt worthwhile and meaningful and we were motivated to start researching and brainstorming solutions.
Most importantly, early treatment with targeted antibiotics is crucial for complete recovery (WHO, 2019). Diving into more and more details about Buruli Ulcer, we’ve found several existing approaches to detect Buruli ulcer, which may also rely on the specific detection of Mycolactone. In fact, this approach has also been suggested by the WHO and previously followed by a group of researchers who exploit that relation in thin layer chromatography (Pluschke and Röltgen, 2019).
Yet, these techniques all require well-equipped labs, well-trained health workers and time. There is a standard method based on PCR to confirm suspected Buruli ulcer cases, but this methodology requires relatively long time and a well-equipped laboratory (WHO, 2019). The necessity of reliable, cheap and easy detection methods crystallized as a major priority in scientific literature and on behalf of the WHO.
After an extensive literature research and an exchange with Dr. Kingsley Asiedu from the WHO about the usefulness and feasibility of the new approach, we finalized a first draft of our new diagnostic tool.

1.2 A briefing on the general principles

The working principle for the detection of Mycolactone in our new approach is based on the conformational change of an RNA-structure called aptamer, which can serve as a switch and can be turned on or off if Mycolactone is present (Sakyi et al., 2016). Our search thus further centred around detecting Mycobacterium ulcerans based on the specific interaction of this toxin with the aptamer which we introduced as a riboswitch into an E. coli reporter strain, further referred to as GEM (Genetically Engineered Machine). Over these past months, there were several questions which we had to answer as regards the realization of this basic idea, its usability, its feasibility for the intended use, its safety and ethicality, and its position in the broader context. This is why we have attributed a considerable amount of time to integrated Human Practices.

1.3 Stakeholder analysis

We started to think not only inwards – about the genetic switch and our GEM – but also outwards – about how our GEM would be applied, where, in which context and environment, and especially, by whom. These considerations were to help evolve, stir and control our project further.
To this end, we brainstormed stakeholders who may be affected by our new test kit. The results of this early brainstorming are shown in Figure 1. Stakeholders with a yellow halo have been identified as of particular interest. This was either due to our anticipation of their provision of valuable information for our project, or due to the direct effect our new, ready-to-use test kit may have on them.

BOKU-Vienna--HumanP-Grafik1.png
Figure 1: Stakeholder analysis by our team. Yellow halo signifies those who we focused on when reaching out for a dialogue on our project.

Hence, representatives of these stakeholders have been contacted throughout our project and we have been in vivid dialogues with diverse people from the very beginning of our project. The detailed outcomes of these interactions are presented in Part B of this paper, mostly in the form of interview summaries. The following sections will refer back to these summaries to demonstrate how they have stirred, guided and improved our project.

2. The three layers of our project progress

Based on the input from the stakeholders mentioned in 1.3, as well as extensive literature searches and exchanges with our Principle Investigators (PIs) and others, our project has evolved from a rough, preliminary idea into a detailed plan and actions.
This section will serve as a brief summary of the progress of our iGEM project on three levels: The GEM (Genetically Engineered Machine, our reporter strain) level, test kit level and the level of integration of our tool into other disease mitigation efforts. This structuring of reporting progress is represented in Figure 2 for an overview.

T--BOKU-Vienna--Integreated_HP_Figure2.png
Figure 2 The three layers of monitoring our project progress and their interdependencies.

The following sections outline the development of our project on each of these three levels, from the most preliminary idea to the final results, and showcase suggestions for future follow-up activities.

2.1 The GEM level

2.1.1 First genetic switch design

After coming up with our broad idea – to diagnose Buruli ulcer over Mycolactone with a GEM – we looked into several ways of solving this issue at the molecular level. For that, we screened through literature and parts which have been used by other iGEM teams around the world. Eventually, we came up with a first idea for our GEM, a genetic construct and our test kit [see Project Description on WIKI].
Right from the start, we contacted Aptagen, a company specialising in aptamers for analytical purposes. They kindly provided us with aptamers specific for Mycolactone and some expertise insights into the work with aptamers. The support they offered was useful for the whole range of experiments our wet-lab team conducted.

2.1.2 The idea to replace the Mycolactone riboswitch with other riboswitch

Also, due to this exchange with Aptagen and their insights, we got an estimation of how easy it may be to adapt our construct to other diseases as well, as the aptamer in the riboswitch is an easily adaptable part. Such a switch may be adapted to signature substances of any disease. The screening with Aptagen for potential targets and the Selex would always be possible.

2.1.3 Switching from Mycolactone to theophylline

The first rounds of experiments did not go very well with the Mycolactone riboswitches. For the reasons above and our understanding that we may easily change to another aptamer system, and that the construct behind the riboswitch alone may already be of greater value, the wet-lab team switched to a theophylline riboswitch which is much better characterized. This was aimed at generating a proof of principle for the workability of the construct downstream the riboswitch and serves as one example where an idea which came out of an exchange with experts was implemented into a the GEM design.

2.1.4 The decision to try cell-free systems

However, the wet-lab team reported repeatedly reported modest performance of the Mycolactone riboswitches in the first few trials in a cell-based system. This is when the team started to think about alternatives, and found out about cell-free systems. We were lucky enough to find Arbor biosciences, a company providing high-quality cell-free system kits. And we were even more fortunate when we received their kits as a sponsoring. That meant that we did not only have the material, but also a partner to turn to with questions on cell-free applications.
After Dr. Nichter also mentioned a cell-free system as an alternative for GMO-based systems, we decided to look further into cell-free systems as an alternative setup of our test kit. When Arbor biosciences generously sponsored us with a test kit for cell-free testing, we started planning how to implement it in our system.
The main considerations for switching to cell-free systems were further based on safety considerations. One of the main hazards which we identified in our cell-based system was the antibiotic resistance markers, which could be transferred to the environment if the GEMs were not disposed of correctly or not handled in a way which prevents any release into the environment.

2.1.5 Cell-free system for riboswitch screening

Also, to screen for feasible riboswitches, a cell-free approach appeared more than worth a try. After thorough discussions with Dominik Jeschek, a cell-free expert, considerations of the detailed manual provided by Arbor biosciences and extensive literature search, the wet-lab team set up an experiment to screen for the best construct. The cell-free system here was a quick alternative to the longer, cell-based procedure. This helped us to identify the least leaky and most specific Mycolactone riboswitch, which would now be incorporated into living systems in the next steps. For more on this, check our Results .

2.1.6 Choosing cell-based over cell-free for our kit

Yet, another talk with Prof. Dr. Reingard Grabherr made us reject the idea of using cell-free systems in our test kit. Even though cell-free systems are extremely useful tools for screening in laboratory settings, they may not be useful for the purpose we aim for in field applications. For the particular purpose of trying to make a test which is easily accessible, easy-to-use for anyone and cheap, a cell-free system may not be the optimal option. It requires an intact cooling chain, some more sophisticated equipment and is significantly more expensive. This means that, even though it was very useful for the purpose of testing our constructs and screening for an optimal riboswitch (see: Results), its disadvantages eventually outcompeted the considerations for safety. It is worthwhile to emphasized here that we learned that the design process needs to strike a balance between safety, applicability and cost aspects. In this case, the benefits of a robust system based on E. coli did outweigh those of a cell-free system for an application in our own test kit. Also, our interviewees such as a former patient Paul Anejodo, or experienced medical expert in Togo, Dr. Maman Issaka, as well as Dr. Kim Blasdell from CSIRO and Prof. Dr. Reingard Grabherr agreeing that they would not see many problems with GMOs in the test kit, as long as basic safety measures are considered. This was also the main point by Martin Moder who argued that GMOs for medical purposes are widely accepted, and in our case even more so, as the risk-benefit ratio is high and the GMOs do not come into contact with the patient. Hence, the final decision fell on the use of cell-based systems over the use of cell-free ones.

2.1.7 Re-designing the genetic construct downstream of the switch

Unfortunately, there have been many problems in the implementation of the test kit as it had been designed in the beginning of our project. The enhancer mechanism over LuxI and LuxR and AHL did not work well. Only T7 promotor and T7 polymerase worked in the constructs. Likewise, the yellow chromoprotein which was originally meant to serve as viability control under the constitutively expressed Arabinose promotor did not give the clear visual readout we had hoped for. However, we wanted to provide a strong read-out of the viability control. This is why we were forced to look for alternatives. The part which was chosen by the wet-lab team, however, did not entirely match these requirements as a green fluorescent protein, GFP, was chosen. This was for reasons of availability. We are, however, aware that this impacts the accessibility of the test as a fluorescence photometer is needed to check for viability. This is further discussed in the sections on the test kit procedure.

2.2 The test kit

Beyond the molecular technicalities of our GEM, we have been thinking about how to integrate this GEM in a workable test kit. In this endeavour, we have referred back to our results in the considerations of our stakeholders, have defined our users and their environment based on literature search and interviews, and have readjusted our design based on the feedback from stakeholder interviews, lab results and literature searches.
It is worthwhile to emphasize here that this is a solely a draft of a theoretical, virtual test kit assemblage. We have not managed to create a workable GEM with the whole genetic construct therein during our time with iGEM. However, should this project be continued in the future, we suggest the following propositions.

2.2.1 Potential users

The users had to be defined in order to consider necessities and requirements in the design of our test kit. In the beginning of our project, we had not clearly defined the users of our test kit, and partly also considered making the test available to patients at the supermarket as well for self-diagnosis. Nevertheless, the main focus had always been on making them available to point-of-care health workers. Once the design at GEM level became more defined, we discarded the former idea due to safety concerns. At latest after the interview with Prof. Dr. Reingard Grabherr, who brought to our attention that the test kit must be used by trained personnel only, we narrowed down our end users. It should be emphasized here, for this reason, that training will also be essential before any kit may be used.
Based on our brainstorming, we identified health care workers on all different levels as potential users of the test. This may include field workers and point-of-care health workers in particular, as well as health care workers in established laboratories, national laboratories and hospitals. Based on the interview with Kim Blasdell, we also identified researchers as potential users if we manage to make the test so selective that Mycolactone detection in other matrices, and hence the kit may be used as a fast test in transmission route research.
Based on what Kim Blasdell said in the interview, and what we had also found in literature, it seems that the main issues with lack of workable detection and access thereto are prevalent in countries in Africa. While relevant laboratories in Australia have better access to certain basic equipment, we cannot expect this according to Dr. Maman Issaka, Paul Anejodo and Emmanuel Agumah in many affected regions in Africa. In the interviews with the latter three, we have tried to get to the bottom of what equipment and infrastructure is available in the field for field health workers and small medical facilities. The answers were sobering, and we cannot assume that even the most basic equipment is always available in these regions.
We also defined that the cooling chain may not be easily kept upright in many of our target regions, affected regions in rural Africa and the Americas. This was considered in the further design of our test kit.
As a test kit is, however, only workable if accessible for all users, we defined this as status quo and decided that our test kit just comprise anything which is needed in the kit, ready to use for the point-of-care workers in the most unfavoured regions.
Having defined our users and their environment, we then considered the three main aspects of our project at test kit level: The test kit deliverables, the operating procedures and the manual.

2.2.2 The components delivered with the test kit

Since we defined that our “worst case” users do not have access to equipment nor basic infrastructure, we decided that we would provide all materials and chemicals with our test kit, as well as an instruction manual. Results .
This is why we have tried to brainstorm any and all materials necessary to be included in the test kit. This, of course, comprises the functional centrepiece of our test kit, namely the media, the GEM and the sampling devices as well as other equipment which may be provided on request.

Freeze-drying

For the test itself, we have decided to provide the medium in a freeze-dried form, and deliver the freeze-dried bacteria in a vial with a septum cap. The freeze-dried medium can be easily re-dissolved, and the freeze-dried bacteria may easily be re-activated therein. This is to circumvent problems with discontinuities in the cooling chain. The 1 M sodium hydroxide solution was also provided as we may not expect our test kit users to else have access to this chemical in the field.

The containment of GEMs

The septum lid was an idea based on the interview with Prof. Dr. Grabherr, who suggested to keep the vial with our GEMs as closed as possible. The septum thus may be penetrated with a needle to add sample, medium and inactivation liquid. No further opening of the vial takes place at any point in the procedure, and the risk of release into the environment is minimized.

Fluorimeter

A major bottleneck in the design of our test is the viability control of our GEMs producing green fluorescent protein (GFP). Fluorescence can only be measured by a fluorescence photometer, which, of course, is seldomly available in the field. Therefore, we have looked at other iGEM projects who have run across the same issues and have found the fluorimeter designed by iGEM team Aberdeen 2014. They constructed a cheap, portable Raspberry Pi computer-controlled fluorimeter suitable for developing countries which only costs around $100 (iGEM Aberdeen, 2014). We would consider adapting this device for our purposes and sending it along. Or, even easier, we would include a small UV flashlight as no quantitative measurement of fluorescence in necessary, but rather the mere presence and absence may be sufficient to check whether the GEMs are viable.

Consumables

For the procedure described below, we would also include sterile swabs for the sampling, and the needles and reservoirs necessary for fine needle aspirates. Further included would be sterile useable, as well as the needles to transfer medium, sample and inactivation solution into the contaminated vials via the septum. This has been decided as Emmanuel Agumah told us that no autoclaves are available for sterilization, and ethanol for disinfection may not be suitable or not available for all purposes needed.

A field centrifuge

Also, this device may not be available for health care workers in the field. To mitigate this, we have searched in the iGEM project database for a solution to this issue and have found a 3D-printed hand-powered centrifuge by Byagathvalli et al. (2019), which was designed as part of the Lambert iGEM 2018 team’s project. This light and easy-to-use centrifuge is cheap and can be added as a test-kit component or on request.
For a schematic workflow of the test kit operating procedure see Figure 3. Please find a detailed list in the Results section of our WIKI under Test kit and manual.

2.2.3 The operating procedure of the test kit

Based on considerations of the potential users of the kit and their access to equipment and infrastructure, we drafted the test kit procedure and the respective manual.
While considering the working environment and the ideas that the test shall be easily performable outside the lab under field conditions, we deliberated on the following points.

Figure3
Figure 3: Schematic depiction of the procedure of our diagnostic test. The dried medium (MD) are dissolved in sterile water (H2O), then added to the reporter bacteria (GEMD), incubated (GEM) and to the patient sample (S). Incubation steps include pre-incubation prior to sample application for 3 h at 37°C, and 2 h at 37°C after sample addition. Samples are spun down and compared to a color code to distinguish negative from positive samples.
Critical steps

We tried to minimize by design such critical steps in that procedure which would render the procedure less accessible for field workers. After designing the procedure, we defined the steps which may require most attention within this procedure.
Again, one critical step to mention is the use of a fluorimeter which may not be available in the field and requires electricity to be operated. We hope to circumvent and mitigate this limitation by providing a cheap and reliable mini-fluorimeter, such as the one developed by iGEM Aberdeen (2014).
We also considered that there may not be any incubator available for the incubation steps and hence included a table where alternative temperatures may be applied, with alternative time regimes, as suitable to the outside temperatures to as low as 30°C in an given situation, so that the bacterial suspension may be incubated outside in the sun or similar. Another option may be a portable incubator.
Also, the centrifuge to spin down the samples may not be available at the point-of-care, and should be provided. For instance, the model by Byagathvalli et al. (2019), which was designed as part of the Lambert iGEM 2018 team’s project is an option.

Training

We are aware that training will be of utmost importance for these procedures, but considering the users being field workers who need to be able to take samples, we can expect that they will need at least some prior training anyways. Especially fine-needle aspirates should only be taken by experienced health workers (Asiedu, 2014).

The need for confirmation

After the results are registered and documented, a confirmation may be necessary according to national Buruli ulcer programs (i.e. 70% of all cases shall be confirmed by the gold standard PCR, 100% are in theory confirmed in Australia according to Kim Blasdell). This means that our test kit would certainly not serve as a replacement for the confirmation via PCR, but may significantly lower the times until results are obtained. The latter has been named as a major bottleneck by Dr. Maman Issaka and others, which our significantly faster test kit which may overcome if applied at the point of care.

Quality control

Thinking forward, a validation and quality control of our test kit are imperative, and it will show how reliable the test is and how powerful it is with regards to false negatives. Such a validation may be done against PCR, similarly as in the paper by Wadagni et al., 2015. The results of such a validation study would then further require consideration of the respective follow-up actions. However, this was beyond the resources of this project and may become relevant only if the project is followed up.

Disposal and waste management

One thing which definitely deserves high attention is the disposal of the used equipment and reagents, as they contain living GMOs. We have discussed several options for this, for instance kill switches or auxotrophy markers, which may in the future be introduced into our GEM.
However, considering the time left and the application of our test, the most feasible seemed to be the addition of 1 M sodium hydroxide solution (NaOH) in a 1:1 dilution and incubation for five minutes before disposal which was suggested by Prof. Dr. Grabherr. We have indeed tested the efficiency of 0.5 M NaOH on the E. coli strain we are using and have shown that a significant reduction in CFU is reached (Figure 4).

Figure 4
Figure 4: Top two sixths: 0.5 M HCl; left and right sixths: control; bottom two sixths: 0.5 M NaOH. Incubation for 5 minutes prior to plating on PCA.

Of course, we are aware that this purely optical reduction would need to be proven by many more replicates, and that there may still be a chance of single survivors. Hence, as discussed in the safety by design section, a model with multiple barriers should be established. We have discussed this with Prof. Dr. Grabherr as well as Martin Altvater, who have further suggested to burn the potentially contaminated useables for a thorough removal and inactivation of all living GEMs, any potentially present Mycobacterium ulcerans and other contaminants in the patients’ sample. This would also destroy and inactivate the antibiotic Kanamycin which is added to all media used in the test. The instructions concerning the safe disposal shall also be an integral part of the manual and users shall be trained for this procedure. This approach has also been endorsed by Emmanuel Agumah.

Legal provisions

Legal provisions shall always be considered. Depending on the target country of use, these provisions need to be studied carefully and considered at all stages of delivery, application and disposal, with special regards to requirements for the contained use of GMOs.

2.2.4 The instructions manual

An integral part of our efforts is certainly the instruction manual, which shall be designed under considerations to several factors. We have not managed to compile a detailed manual due to the fact that the GEM and further, the test kit design are both not final yet. Still, we would consider three main criteria while writing this manual based on what we have learned from experts (i.e. Dr. Kim Blasdell, Dr. Mark Nichter, Dr. Issaka Maman) and literature search. The draft manual has been designed according to the following considerations and can be found under Results.

Understandability and Clarity

Firstly, it needs to be understandable for all users, despite the fact that many affected regions have several national languages. Paul Anejodu has also offered his help in translations, and we have also suggested to use pictograms in order to maximize readability and ease of use. This would be the next steps toward improving our manual.

Usability

Secondly, as explained on our project inspiration and design page, the usability of our test kit strongly depends on the fact that it can be applied easily without extensive training and equipment. Yet, to ensure a reliable performance of the test, as well as safe containment and disposal of our GEMs, clear and detailed instructions which are easily understandable are crucial for the functioning and usability (see also: safety by design considerations, chapter 3).

Completeness

All steps must be covered in the manual, so as to minimize uncertainty with the end users and indeterminacy, leading to unintended use. This should be evaluated as well. However, our version does not claim to be perfectly complete yet and would require evaluation with field workers in a continuous feedback cycle.

Reference to Standard Procedures

The manual should not only refer to other standard procedures (i.e. patient sampling procedures), but also include the information on these procedures instructions in the manual at hand for the convenience of the user. The reader is referred to the manual by Portaels (2014) for the WHO for more details, which is directly cited in our draft manual (accessible over .

Evaluation and Improvement

As the three criteria mentioned above are essential for the value of the test kit manual, it seems worthwhile to organize an evaluation with potential end users. We suggest making the manual available to health care workers in different endemic regions and ask for their feedback. We may send along a mock test kit (without our GEMs included) to see if the handling may be improved. Altogether, the manual shall be revised continuously so as to ensure that the quality of the kit and its safe and reliable application is always adapted to new circumstances and improved based on experience and best practices.
Aspects of the evaluation process shall be easiness, clearness, completeness, feasibility and sources of errors. This would also help with regards to the issues raised in chapter 2.3, where the absolute necessity to think beyond technical issues is discussed.

2.3 Integrating our tool with other efforts in disease mitigation

While it is true that the facilitated detection of the disease will add to the improved treatment of patients according to experts (i.e. according to Dr. Maman, Dr. Asiedu and Dr. Blasdell) and others, there is a third layer which needs to be considered. Solely making available easy-to-use kits may not solve the problem of underestimation and lack of knowledge and awareness amongst affected communities. This has been extensively discussed elsewhere (i.e. Plüschke and Röltgen, 2019), and we were inspired by Dr. Mark Nichter and Emmanuel Agumah who are working on raising awareness for the disease. The following sections show the results of our brainstorming for future measures, inspired from a compilation of efforts outlined by Plüschke and Röltgen (2019).

2.3.2 Raising awareness

For a disease like Buruli ulcer, which is often linked to stigmatization of patients, the communication of the disease amongst health workers is a crucial part for mitigation and early detection (Nichter, 2019). There have been worldwide efforts for the raising of the awareness of the disease amongst health workers in endemic regions over the past two decades. For instance, the Global Buruli Ulcer program, and the BUVA (Buruli Ulcer Victim Aid) and the ABURA (Achieving Buruli Ulcer Reduction and Awareness) associations have shown successes in terms of raising awareness. Also, Dr. Kim Blasdell stated that the awareness had increased over the past years in many areas in Australia.
Moreover, there may be large numbers of undetected cases due to the lack of effective monitoring programs in place. There may even be a systematic underreporting of cases, involving deaths, according to Dr. Anejodo. Raising awareness is an important tool to enhance the chance of the Should we follow up in this project, we would certainly feel responsible for awareness-raising campaigns in addition to training programs. disease being detected at an early stage and curbed further down the line.

2.3.3 Educating and Training Health Care Workers

What our test kit does not solve, but certainly depends on, is the ability of health care workers to take samples from suspected patients. This requires a certain level of the health care worker, and means that further training programs are crucial. The test kit may not be of any value for early detection if it is eventually not going to be applied at an early enough stage and if it is not carried out appropriately. Therefore, visiting health facilities and establishing strong relationships with them for future exchanges would be of high importance for further success.

2.3.4 Investigating the spread of the disease

The contraction mechanism of the disease is still not well understood. Dr .Kim Blasdell has mentioned that there are applications beyond the diagnostics. In fact, the test kit could also be applied as a relatively quick and easy alternative to PCRs and cultivation. This would certainly be an area where we may consider getting active as well. At the same time, it is important to communicate that there are certain areas of higher risks, which may be a problem for tourists in Australia according to DR. Kim Blasdell.

2.3.5 Better facilities for in-patients

Dr. Paul Anejodo confirmed in an interview what has been stated in literature as well – there are problems with facilities which make the experience very unpleasant, especially when long-term hospitalization is necessary. There may be a problem with funding and attention. So, tightly linked with raising awareness, there may be an improved awareness for the improvement of health care facilities. An example of a study trying to implement exactly this would be Amoussouhoui et al. 2016 which reported improvements in patient experiences after responding to feedback on bad patient experiences.
Altogether, such efforts may involve targeted information events, games and workshops at kindergartens and schools, appearances at public events and the

3. Safety considerations

This is linked to the concept discussed in the second layer, namely the test kit in chapter 2.2. As explained there, we have thought about measures to prevent the release into the environment of the GEMs as GMOs before, during and after the use of the test kit, so as to prevent potential risks associated with the reproduction outside of the contained use. This inactivation functions on the basis of addition of sodium hydroxide solution which kills the GMOs, and a second barrier where also the antibiotic Kanamycin and the resistance markers are destroyed by burning in a wide pit.
However, as discussed by van de Poel and Robaey (2017), a multiple-barrier concept may be desirable, as well as mechanisms which prevent the release from a contained use.
However, we have not planned to introduce a kill-switches, autotrophy markers or others. And this was not only due to lack of time. In an interview with Prof. Grabherr, we have learned that also these kill-switches may not provide a sufficient safety valve for preventing any and all introduction of GMOs into the environment. This is because there may be spontaneous mutations in that switch design. So, given the number of bacteria in one flask (planned: around 109 CFU/mL), there is a significant chance that bacteria mutate spontaneously and are no longer prone to cell death by activating the kill switch.
As for an auxotrophy marker, we have considered applying one in our construct. However, we simply lacked the time to go about this issue and have left this as a potential option for future improvements of our GEMs.
Instead, the most feasible option for us lays in the design of our test kit components, such as burst proof culture vials, protective packaging and a manual which ensures the safe handling of the test components, and within the scope of the declared purpose only.
At this point, it also seems worthwhile to mention that we consciously reject the approach of trying to achieve an “idiot-proof” design (van de Poel and Robaey, 2017), which aims to design out any and all risks arise due to indeterminacy. Rather, we believe that it will be much more likely to control hazards by adequate training of those performing the tests, hence, by involving people along the usage chain and transferring responsibility on them. We do not want to take the responsibility off the handlers of the test kit, who are, in our case, experienced health workers. We trust that they will follow the procedures and also react to unforeseen events in such a way that risks are mitigated. Also, there should be a frequent revision of the manual and test kit for continuous improvement causes. Yet, we recognize our responsibility as designers as well.
We have discussed this issue extensively with Prof. Dr. Grabherr, who is delegate of the Austrian Commission on Genetic Engineering. With her, we have gained some great insights. She reinforced us in the decision not to claim being able to design out any and all risks, as this is simply impossible due to indeterminacy and uncertainty in general. Rather, what she suggested was a containment of our GEMs which does not need to be opened at any point in time and can even be inactivated “in place” by addition of sodium hydroxide to the sample and GEM suspension after use to inactivate the bacteria.
However, NaOH does not break down the DNA to such a degree that the antibiotic resistance marker found on the plasmid backbone of our GEM is destroyed. This means that the genes may still be transferred into environmental organisms after disposal and release into the environment. Therefore, we have thought about another physical macro-barrier, namely burning the whole waste including the bacterial and sample suspension inside for full degradation. Burning in an open pit removes and degrades the antibiotics contained in the media and any biological agents potentially present in patients’ samples.
This may be particularly feasible for applications in the field, where no autoclave and no kettle, and no waste disposal systems are in place. According to an interview with Paul Anejodo, this is often the case in rural areas, so these restrictions need to be considered, and this approach definitely seems feasible to him, Martin Altvater and Prof. Dr. Grabherr. This was why we believe we have sufficiently considered safety risks incumbent to our kit, established safeguard measures and have designed it to be fit for application in the field.

 

Part B : Interview Summaries, Lessons Learned and Project Integration

  4. Dialogues which have shaped our project

From the very start of our project, we have had the chance to interview several experts in their respective fields. From specialists in Buruli ulcer research, to awareness-raisers for the disease, over former patients to aptamer experts and bioethicists. The information gathered in these exchanges has had a large influence on our project and the way we have been thinking forward. In the following section, you will find the summary reports of the interviews we have conducted and a respective explanation of what we have learned from these dialogues, and how this has helped us to rethink and design our project over the course of the project duration.
Our interviewees have been approached based on an early brainstorming session in which we identified the stakeholders of our project (see Figure 1, Part A).
Hence, representatives of these stakeholders have been contacted throughout our project and we have been in vivid dialogues on crucial info with diverse people from the very beginning of our project. The detailed outcomes of these interactions are presented below to showcase the cruciality of exchange as a starting point for stirring, guiding and improving our project.
Disclaimer: All interviewees, regardless of the means of transfer (via e-mail, skype, phone call or in person) have been informed about the content of the interview, the free choice to participate or terminate participation at any time, the purpose of the interview and what their answers would be used for. All interviews summarized here have been conducted and published with prior informed consent. The interview design was inspired by Jacob and Furgerson (2012) and a Harvard University Guide provided by the iGEM Headquarters.

Dr. Kingsley Asiedu

Dr. Kingsley Bampoe Asiedu is from the World Health Organization, manager of the WHO global Buruli Ulcer Initiative. From the very start, we have been in contact with him several times to update him on our progress via newsletters and ask him for his input. His confirmation that there is indeed an urgent need for better diagnostic tests was all we needed to reconfirm us in our endeavour to start our research project on Buruli ulcer diagnostics. Indeed, he reassured us that we are on an interesting track when he generously provided us with some of his Mycolactone, a very rare commodity as we had to experience. He was also so kind as to have a look at our safety sheets.

Lessons learned and integration: The need for Mycolactone-based diagnostic tests is still high, and rapid tests can indeed mitigate the long-term effects of the disease and make healing significantly shorter. It is worthwhile to consider our approach and also exchange with other people in the field about it who may be interested in following up on this project.

Thomas Caltagirone and Albert Liao from Aptagen

Thomas Caltagirone, PhD, CEO and President at Aptagen, and Albert Liao from Aptagen provided us with their insights into aptamers as advantageous tools in diagnostics, and for the offer to support us in improving our concepts and with trouble-shooting.
He answered questions concerning the advantages of aptamers over, for instance, antibodies. Aptamers offer extreme specificities, high affinities even for difficult targets and small molecules. They are also lower in cost than antibodies and more stable, also to thermal stress and have a longer shelf life.
Further, he offered to help us with some troubleshooting on the Mycolactone riboswitches which they had kindly provided us with. In particular, he suggested we first develop the constructs around the aptamers and then determine the signal-to-noise ratio to determine which riboswitches with which aptamers works best.
He also gave us an estimate of the costs on creating an aptamer/effector switch at know affinities, as well as the duration the company would ask for to come up with a solution.

Lessons learned and integration: Aptamers have many advantages over antibodies, and our approach may add to the range of useful applications. They have provided us the Mycolactone-specific aptamers for our experiments with riboswitches and helped us with screening, i.e. finding balance between specificity and leakiness. The way we are thinking is definitely of worth, as also the aptamer may be exchanged any many other things may be measured.

Dr. Dieter Moll

After presenting our current state of the project to Dr. Dieter Moll from Biomin, we received some valuable feedback on our communication and justification of our project. He questioned whether there are arguments to develop such a test kit if the manifestation of the symptoms is so obvious and characteristic. We took this as an input to emphasize this in questions we have posed to other interviewees down the line and in our literature search.
Lessons learned and integration: It made us come up with better arguments and evidence that indeed, such a diagnostic test kit may indeed prove essential for disease mitigation and the best possible prevention of long-term effects. This was confirmed by Dr. Kingsley, Dr. Blasdell and Dr. Issaka in their interviews. Also, we build our argument on the fact that the test shall serve as a method to diagnose the disease as early as possible, so still in the stage where the skin abnormalities are nodules and not ulcers. This made us further rethink the sampling procedure which we may suggest for our test kit.

Dr. Paul Anejodo

Paul Anejodo is a survivor of Buruli ulcer from Ibaji, Kogi State, Nigeria and he has shared some very personal experiences, and has inspiring us with his determination to spread awareness for the disease.
He shared with us how the disease developed for him 18 years ago, starting out as a small furuncle on his knee, which then burst open and made the skin peel off. This led to stigmatization, mainly also connected to the smell and the sight of the wound. In his community, there was not much awareness about treatments of the disease, and he was sent to traditional healers and churches as the ulcers were connected to witchcraft. Also, what shocked us after not reading too much about fatal cases, he mentions several people who passed away due to the disease. Costs have always been a big issue for patients to seek medical treatments of the disease. And the treatment he received took about one and a half years until he was released from the hospital again.
He also states that awareness is still an issue where he is now, so in Nigeria, but also at his medical school in the US. And besides focussing on new tests, also the health care facilities should be better equipped.
Lessons learned and integration: It is of utmost importance to raise the awareness in affected regions. Also, there seem to have been more death cases than we had thought, probably they also go unregistered due to said lack of awareness and the lack of available diagnostic tests. Maybe a rapid test kit could improve this situation and shed light on dark numbers, so as to reinforce efforts if needed. There are many people fighting with Buruli ulcer and it is a disease which affects you for the rest of the life. Raising awareness is one of the most important things to consider, this is once again a reason why we have introduced that category in the three layers of project progress.

Dr. Maman Issaka

Dr. Maman Issaka, holds a PhD in molecular Physiology and Immunology at University of Lomé in Togo. He is the head of the Molecular Biology and Virology Department at the National Institute of Hygiene, Togo, affiliated with the Ministry of Health in Togo, and provided valuable first-hand insights into the current state of disease diagnostics and experiences with affected patients in Togo.
In fact, he has been working on the detection of Mycobacterium ulcerans since 2011, using conventional PCR. His PhD thesis was also on Buruli ulcer, factors including by analyzing risk environmental, molecular and physiopathology factors and how they could explain the high prevalence of the disease in an endemic region of BU in Togo. Since October 2019, they have installed the detection of the Mycolactone at the regional hospital with the national reference center of Buruli ulcer treatment, using the f-TLC (fluorescent thin layer chromatography) method.
He shared with us that, what has also been mentioned by Paul Anejodo and Mark Nichter, stigmatization is still present with empirical or traditional beliefs (witchcraft, bewitchment, divine punishment) which explain why they are seeking traditional healers. He also mentioned that an increasing awareness in field workers would, with high probability, increase the chance of detection of the disease at earlier stages, as most patients come to the hospital now with stage III ulcers (– the stage in which the ulcer already manifests in lesions above 15 cm in diameter (Pluschke and Röltgen, 2019), expl.). Now, he estimated awareness in endemic regions to be around 40-60 %. However, overall awareness seems to have increased over the past years, since the Global Buruli Ulcer program was started in 1998.
He also brought to our attention that the equipment available for health care workers in endemic regions is often very limited to non-existent. He also confirmed that there normally are no confirmations of cases by PCR at community level, and if samples are analysed, it takes about two weeks until results are in. This has led to the fact that sometimes, treatment is started before results are in, when an expert – an experienced clinician – is certain about the disease.
As to costs, he mentions that the program for PCR and diagnosis are free for patients and covered by partners. However, he does emphasize that the travel to and from the point of care is sometimes expensive and may prevent people from following up or seeking help in the first place. He emphasized though that the funding is still very low, which leads to lack of surveillance of the disease, as well as sample transportation challenges.
He also confirmed what we had read in the literature and heard from Anejodo about the equipment available for health care workers. He mentions the lack of equipment, material and qualified personnel as the main limiting factors for better oversight of the disease. In this background, he states that a quick test with minimal equipment will be very useful for community health workers, and will be valuable especially for the treatment of patients as quickly as possible and for the reduction of the time patients need to stay in hospital.
As for Mycolactone, he was so kind as to tell us that the concentrations in ulcers and wounds is normally around the ng level, and they have detected Mycolactone via f-TLC in this concentration range.
As for the use of GMOs, he believes it is not an issue to use GMOs in the diagnostic test kit and just points out to the fact that there would need to be an authorization by the bioethics committee.
The antibiotic resistance markers present in our reporter strain could be circumvented, he says, by using a cell-free reporter. However, altogether, our approach seems useful to him as it may outcompete f-TLC which needs a lab and other standard procedures which require more sophisticated equipment.
Lastly, he also suggested some interesting contacts which we may have contacted had there been more time, namely the focal point of the national center for BU treatment in Togo, district clinicians, and focal points of BU surveillance in the country.

Lessons learned and integration: Our test kit does have a value for workers in the field, and the easier to use, the better it will be as equipment is very limited. Time is also an important factor, which is why we try to design our test kit into a quick procedure, and our wet lab team tested the limits of this as to how long the minimum incubation times must be for acceptable results. He also mentioned cell-free systems, which we had considered at this point and already deemed less feasible. Very important was the range of Mycolactone in the wounds, which stirred the experiments in our wet lab into testing lower concentration ranges of Mycolactone as well. He was also one of the inspirations to look beyond the mere technicalities but also into raising awareness.

Dr. Kim Blasdell

Kim Blasdell from CSIRO in Australia, has taken the time to provide us with detailed and interesting answers to our questions on the current state of Buruli ulcer research and diagnostic methods in Australia. Kim Blasdell is a CSIRO Research Scientist currently working on a Buruli ulcer project.
She let us know that in Australia, most cases are confirmed with the standard rt-PCR method (IS2404) followed by confirmation with another rt-PCR (IS2606), as laboratory access is okay there. These techniques are also used to monitor the spread of the disease to investigate the routes of contraction. In combination with an RNA-based PCR assay, they check for viability in the bacteria. It was very interesting to hear that a tool like the one at hand may also be of great interest to screen environmental samples (plant, soil, faeces, insects) for M. ulcerans and check for viability, as only living M. ulcerans produces Mycolactone.
In addition, she stated that in Australia, many public awareness programs in affected regions have been fruitful, meaning that many people recognize the disease in an early stage so that healing is a much faster process. However, since affected areas are also tourism magnets, many people develop ulcers away from affected areas and may not receive the treatment there. Kim Blasdell also highlighted that general physicians are relatively aware of the disease already in affected regions, and television campaigns have certainly added to the fact that the awareness is increasing. For her, awareness is one very critical factor.
She was also so kind as to share some insights from patients, who also experience the antibiotic treatment as very harsh and awful. The disease also has a lot of emotional sides to it, as it is such a long-lasting disease with several possible complications.
Lastly, as regards GMOs used in diagnostic test kits, she agrees with other interviewees that a contained use of the latter should not be a problem for the purpose we are suggesting.

Lessons learned and integration: The disease is a horrible disease which, however, may do much less damage if detected early enough. This once again emphasises the relevance of our test kit. Public awareness is a major issue in this regard, and a critical component for early detection. This is why we also need to look beyond the GEM and the test kit and think about ways of engaging people in the fields. Research in transmission pathways is still ongoing, and for this, a tool which detects viable M. ulcerans in complex samples may be of great interest. This is why we should think about using our test kit not only for human samples, but try it with other matrices in the future, to assist in better understanding the transmission of the disease.

Prof. Dr. Reingard Grabherr

Prof. Dr. Reingard Grabherr, Head of the Biotechnology Department at BOKU Vienna, and member of the Austrian Commission on Genetic Engineering, discussed biosafety-related issues with us and provided insights into ensuring the safety of a product containing live GMOs.
She discussed safety by design with us and helped us evaluate and reflect in the main safety considerations we have had in what concerns our test kit. Based on a paper by van de Poel and Robaey (2017), we discussed how to best strike a balance between designing out any risks and leaving responsibilities to the users. In fact, Prof. Grabherr confirmed that our approach cannot be made 100% safe as the application cannot be 100% stirred (as with any technical application). Hence, there is indeterminacy. However, the actors along the chain, especially the end users, do have responsibility and will also feel this responsibility as they have to act following a manual. She also brought to our attention that the kit, however, would always need to be used by an expert, hence someone who is sufficiently trained.
What we can do, however, is overcoming the concerns toward a potential release into the environment by providing a brittle-proof culture vial which is never emptied, and the contents of which stay in contained use only. She explained that this vial can be seen as a closed lab per se, but that considerations are necessary as to how to keep it closed and not bring it into contact with the environment or the patient.
She also gave us some inspiration on how to tackle the issue with antibiotic resistance markers: While there are several systems, from her experience, she finds that antibiotic-free plasmid systems are useful. Also, the construct may be integrated into the genome as well, so that it is more stable and does not need the antibiotic pressure that it needs when it is in a plasmid.

Lessons learned and integration: There are relatively strict, but technically easily achievable requirements when working with contained GMOs. Biosafety considerations require to kill all our GEMs, which are in our case class 1, and that would be enough to ensure safety. She inspired us to think of NaOH addition as inactivation step, which we then tested on our bacteria (see Figure 4). Also, she brought us the idea that it would be desirable to integrate some sort of indicator into the medium so as to check for the pH value and hence indirectly control the efficiency of inactivation. We considered this in our test kit design. It was also after this discussion that we came up with the idea of vials with septa, so as to make sure the opening of the vial and exposure to any surfaces of the environment (syringes, vials,.. ) is kept as low as possible. The users should also be clearly defined, and we defined the only feasible way to be trained health workers who will follow the instructions in the manual, and who are able to take the samples from the patients.
While there was no time anymore redesign and integrate our switch from the plasmid on the strain genome, we have certainly kept this as a priority should the project be continued in the future.

Dominik Jeschek, MSc

Dominik Jeschek, MSc, PhD candidate researching in Potsdam, has provided us with valuable insights into practical work and the experimental setup of cell-free systems. When we explained him the plans for our experimental setup, he helped us make the experiments more effective and has finally helped us tremendously with these small, considerate changes.
Also, we discussed with him the advantages and disadvantages of cell-free systems over cell-based systems, with particular respect to our project. In fact, it was him who brought to our attention the fact that the cooling chain and the equipment for detection as well as the set-up of a cell-free system pose significant disadvantages over a cell-based system, even though it is true that our cell-based system has one major disadvantage being the antibiotic resistance marker. Yet, for aptamer switch screening, the cell-free system provides an excellent basis for this purpose, and he helped us design our experiment based on our Arbor biosciences test kit manual.
He explained us very well the particularities of conducting cell-free measurements and argued about their advantages and disadvantages for certain uses. His practical tips have helped the wet-lab team tremendously. In fact, this turn has led to some nice results on the measurement of Mycolactone riboswitches.

Lessons learned and integration: The most important input was the discussion of cell-free versus cell-based systems, which made us recognize the advantages of the cell-based system for field use in the test kit, and those of cell-free systems for aptamer screening, which we then discussed further with Prof. Dr. Grabherr and implemented in our lab work. Our candidate aptamer switches were screened in a cell-free system and one could be selected as most specific and least leaky. It made us focus our test kit design on cell-based systems again.
The GFP measurements, which we had been conducting before, were all relatively unstable, but measuring fluorescence in a black multiwell-plate worked much better. His contributions, tips and practical hints for performing cell-free analyses helped the wet-lab team understand experimental planning and has resulted in some nice results: We could successfully screen for a favourite riboswitch candidate.

Martin Moder, PhD

Martin Moder, PhD, Science Buster, author and molecular biologist, for making the time and evaluating our presentation, discussing science communication and difficulties with public acceptance. He let us know, as an experienced science communicator, that there are little concerns about the use of GMOs in diagnostic tests. Here, GMOs are much more accepted than in applications with direct intake. He also elaborated on the discussion in which people are scared of things with small risks, because there are any risks at all, but do not sufficiently weigh the risks against the benefits in comparison to the status quo. This means that the application of biotechnology, and synthetic biology in particular, should always be assessed by weighting the benefits against the risks of the already existing approach or status, and not against zero risks. The communication of this is crucial for the discussion of new techniques.

Lessons learned and integration: The different types of biotechnology have very different perceptions amongst the public. Agricultural biotechnology is by far more contested than medical applications or industrial ones. The key is always communication and raising awareness with facts. Knowing how to communicate science is a great asset to have. The right way of communicating the project may play a role in increasing the awareness. Generally, telling a story and including provocative pictures for depiction of the cruelty of the disease, less text, more pictures and a clear storyline would make our presentation more effective.

Mark Nichter, PhD

Mark Nichter, PhD, University of Arizona, discussed with us some social science aspects of Buruli ulcer research and diagnostics. He has organised himself co-authored several publications on how to increase awareness and set up community Buruli ulcer outreach programs in affected areas, which have inspired us to look beyond the mere technicalities of our test kit.
He made us aware of the fact that many people in rural areas do not have access to “western” medical institutions, but do believe in witchcraft and faith healers as well, mainly due to lack of awareness and infrastructure.
Also, he brought to our attention that one must be very careful with the term stigmatization in relation to the disease, as it can mean different things. He thinks that stigma has been overplayed in many cases, and that the faster the diagnosis and treatment, the less stigma is created. Much of stigma comes from the sight and smell of the wound, he said. Generally, stigma is worst for children. Also, there is a fear of witchcraft which exists, but he stated that this is not so much of a problem among those treated and cured. In general, he shared with us that women tend to be more open to treatment and diagnosis, if the costs are low enough. For children or men, this is worse.
As for the use of GMOs, he believes that it is a matter of establishing trust for GMOs in the local population. When it comes to body fluids, there may be some concerns with the local population, but it would require a survey (which he suggested to set up, but did not suit into the time frame of iGEM 2019 for us) to answer this research question.

Lessons learned and integration: Based on his input, his publications and other literature, we became aware of the importance of not only providing a technical solution, but also implementing it in and for society. The ways this may be done are manifold, but interdisciplinarity and building a trust in science may certainly help in increasing awareness and mitigating the disease. Also, the potential scepticism about bringing body fluids into contact with GMOs provided one more argument for considering a cell-free system.

Prof. Dr. Michael Sauer

Prof. Dr. Michael Sauer from BOKU Vienna has introduced us to Human Practices as a major part of the iGEM project and has done an introduction to bioethics. He has also assisted us with considerations on ethics report and checked on our considerations and essay which had been available so far. There are many ways to consider ethics in a project in synthetic biology. Diverse approaches may lead to diverse outcomes as to whether something is acceptable or not.

Lessons learned and integration: The outcome of our ethical analysis was based on these deliberations and discussions. Therefore, we have come to the conclusion that it is ethical to use GMOs for the purpose of a diagnostic test when these are maintained for contained use, and given that the safety barriers to prevent contamination and release into the environment beyond the use, and the disposal.

Emmanuel Agumah

Emmanuel Agumah is the Director at the ABURA (Achieving Buruli Ulcer Reduction and Awareness) Foundation and President and Founder of the BUVA (Buruli Ulcer Victims Aid) Foundation and is based in Ghana. He has provided us some replies as regards the current practices of waste and post-experiment sample disposal.
We have discussed with him whether NaOH addition for inactivation of our GEM would be suitable, if the current situation of point-of-care health workers is considered. He informed us that the current practices at small health facilities only involve the disposal of any material which is potentially contaminated in pits, but no sterilization is performed, simply for the reason that no autoclaves are available. At District Hospital level or higher, sterilization is performed.
When we discussed another idea which we had gotten when brainstorming safety by design, namely burning the samples, he said that this may make the situation much better, especially when GMOs are then present in the samples, and he suggested to simply do this in a wide pit which should be available at any point-of-care facility. In addition, he suggested to use both NaOH addition and burning as this would create a two-way inactivation of our GEMs and potentially present Mycobacterium ulcerans.

Lessons learned and integration: We learned that the disposal cannot rely on any sophisticated equipment when it comes to small health facilities. Since these are the targets of our easy-to-use tests, we have considered these limitations in the design of our test kit.

  5. References

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