Difference between revisions of "Team:Oxford/Chagas Disease"

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     <h1>Chagas Disease</h1>
+
 
 +
     <h1>Applied Design</h1>
 +
 
 
     <h2>Introduction</h2>
 
     <h2>Introduction</h2>
−
     <p>Chagas disease is a neglected tropical disease named after the Brazilian scientist, Carlos Chagas, who first described the life cycle of the parasite – <em>Trypanosoma cruzi</em> (<em>T. cruzi</em>) that causes the disease.  Chagas is primarily transmitted via the faeces of triatomine bugs, when they take a blood meal. Other forms of transmission include: blood transfusions, orally via ingestion of contaminated fluids, vertical transmission. Chagas is endemic to Latin America but increased migration of infected people has led to it spreading to non-endemic countries, consequently increasing the number of people susceptible to the disease and causing it to be a growing global concern. </p>
+
     <p>
−
<p>Since the 1990s, strategies to reduce the impact of Chagas in endemic countries have largely focused on preventing transmission through vector control programmes and screening blood banks. Although these achievements have significantly reduced its incidence, they  are not sufficient to  combat the spread of the disease vertically from a mother to her child. Therefore, congenital Chagas disease is growing in epidemiological importance, as it is now one of the most persistent forms of the transmission among the human population, with prevalence in some rural areas of Bolivia being as high as 70.5%.  <br /> <br /><br /> </p>
+
    iGEM encourages all teams to take their projects beyond the lab and to consider design using a holistic approach. The question “What is our real world problem?” has been a key consideration from the beginning and has guided our project throughout the summer.
 +
    </p>
 +
    <p>
 +
    To ensure we were putting our diagnostic device into context, we considered various aspects including safety, accessibility and socioeconomic factors in Latin America. Various design iterations were built upon over the course of the summer, influenced by discussions with experts from a range of disciplines - including blood coagulation, microfluidics and general diagnostic devices.
 +
    </p>
 +
    <p>
 +
    Having examined these design options more carefully, we propose a final design for our system which fulfills our criteria for a suitable diagnostic device.
 +
    </p>
  
−
<p>All life stages of <em>T.cruzi</em> secrete a specific protease, known as cruzipain, which allow the presence of the trypomastigotes to be detected by our biosensor. However, the levels of trypomastigotes in the human blood falls with time after infection, as shown in figure 2. </p>
+
    <h2>Developing Our Design</h2>
−
<img class="img-responsive" style="margin-left: -50px" src="https://static.igem.org/mediawiki/2017/4/49/T--oxford--chagas_disease--life_cycle.png">
+
    <h3>Criteria for a suitable diagnostic device: considerations from the OpenPlant Forum</h3>
−
<h6>Figure 2: Lifecycle of <em>T. cruzi</em></h6>
+
    <p>
−
<p>Within 4-8 weeks of being bitten, adults move from the acute phase of Chagas disease to the chronic phase if untreated. </p>
+
    Our early design criteria was influenced by research into the challenges of designing new healthcare technologies in developing countries. Our research indicated that important considerations included the level of infrastructure present, the cost to the end-user and the amount of training required. (http://apps.who.int/iris/bitstream/10665/70543/1/WHO_HSS_EHT_DIM_10.13_eng.pdf)
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/3/38/T--oxford--chagas_disease--evolutions-chart2.png">
+
    </p>
−
<h6>Figure 3: Scheme of evolution of <em>T. cruzi</em> trypomastigotes in the blood of a  human host </h6>
+
    <p>
−
<p>During the chronic phase, cruzipain levels are very low in the blood and cost effective diagnosis of adults focuses on detecting antibodies specific to <em>T. cruzi</em>. However, antibody based diagnosis is unsuitable for newborns who lack a fully developed immune system. Newborns infected with congenital Chagas disease remain in the acute phase for up to 9 months, during which period there is no cost-effective diagnostic currently available. We hope to fill this gap in the ability to diagnose congenital Chagas disease in newborns, using synthetic biology to create a specific protease detection system. </p>
+
    In order to gain an insight into various aspects of synthetic biology, members of our team attended the OpenPlant Forum in Cambridge, UK. Dr Tempest van Schaik gave a talk titled ’Designing Diagnostics’, using her expertise in the development of bench-to-bedside healthcare technologies and importance of the end-user experience. From her talk, 3 key points stood out to us:
 +
    </p>
 +
    <ol>
 +
        <li>Understand your analyte</li>
 +
        <ol>
 +
            <li>What is the context in which the kit will be understood?</li>
 +
            <li>How exactly does taking blood work? Will we transport the blood to a different place, or do a spot-test by the bedside?</li>
 +
        </ol>
 +
        <li>Understand the users of the kit</li>
 +
        <ol>
 +
            <li>How will they be given the kit?</li>
 +
            <li>Will they want to use it?</li>
 +
            <li>Do they want it?</li>
 +
        </ol>
 +
        <li>Understand the diagnosis procedure</li>
 +
        <ol>
 +
            <li>What problems are encountered from an end-user perspective?</li>
 +
            <li>Can we simulate a diagnostics procedure to discover an issues?</li>
 +
        </ol>
 +
    </ol>
 +
    <p>
 +
    Having discussed these findings as a team, we proposed a set of general criteria to guide our initial design brainstorming.
 +
    </p>
  
−
<h2>Symptoms and current diagnosis</h2>
 
−
<p>Diagnosis of Chagas disease is difficult, as the disease is mostly asymptomatic in the acute phase and for the majority of the chronic phase. However, prolonged onset of the chronic phase leads to 30% of patients developing cardiac disorders and up to 10% developing digestive, neurological or mixed alterations that cause 1200 deaths per year.
 
−
The main diagnostic methods currently used to diagnose Chagas are summarised in the table below:</p>
 
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/76/T--oxford--chagas_disease--chagas_disease.jpeg">
 
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<br/><br/>
 
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/7f/T--oxford--chagas_disease--stage_of_chagas.png">
 
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</div>
 
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<table class="table table-hover">
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     <h3>Our 4E’s Applied Design Framework</h3>
−
     <thead>
+
    <p>
−
      <tr>
+
    Applied design is an important component of most iGEM projects, and requires an integrated and holistic approach to ensure projects are considered from a ‘real-world’ perspective. Based on our findings from OpenPlant and research, the Oxford iGEM 2017 team came up with a framework for considering applied design - the 4 E’s (‘Effectiveness’, ‘Ease of use’, ‘Economics’ and ‘Environment &amp; Safety’).
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        <th>Test</th>
+
    </p>
−
        <th>How it works</th>
+
     <p>
−
        <th>Benefits</th>
+
     This provides a structured method for applied design considerations, and we hope that this framework may prove useful for future iGEM teams.
−
        <th>Limitations</th>
+
    </p>
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        <th>Suitable for newborns</th>
+
    <div class="row four-e">
−
      </tr>
+
         <div class="col-sm-3">
−
     </thead>
+
            <div class="four-e-box" style="background: rgb(190,15,52)">Effectiveness</div>
−
     <tbody>
+
            <ul>
−
      <tr>
+
                <li>How long will it take to get a clear result?</li>
−
        <td>Whole parasite microscopy</td>
+
                <li>How will we ensure the test it sensitive and specific?</li>
−
        <td>
+
                <li>How can we ensure it can be used at the bedside?</li>
−
        <ul>
+
            </ul>
−
        <li>Preparation of Giesma blood smears</li>
+
        </div>
−
        <li>Visualised using light microscopy</li>
+
        <div class="col-sm-3">
−
        </ul>
+
            <div class="four-e-box" style="background: rgb(72,145,220)">Ease of Use</div>
−
        </td>
+
            <ul>
−
        <td>
+
                <li>What equipment would be necessary to use it?</li>
−
        <ul>
+
                <li>How can we present the result clearly?</li>
−
        <li>Established method</li>
+
                <li>What level of training would be required for the end-user?</li>
−
        <li>Carried out by already trained professionals</li>
+
            </ul>
−
        </ul>
+
        </div>
−
        </td>
+
        <div class="col-sm-3">
−
        <td>
+
            <div class="four-e-box" style="background: rgb(207,122,48)">Economics</div>
−
        <ul>
+
            <ul>
−
        <li>Not suitable for the when there is little <em>T. cruzi</em> in the blood</li>
+
                <li>What materials will be used?</li>
−
        <li>Not always possible to differentiate between <em>T. cruzi</em> from <em>T. rangeli</em>, which does not cause disease in humans.</li>
+
                <li>Will it be feasible to transport our device in large quantities?</li>
−
        </ul>
+
                <li>How can we alter it to reduce the cost?</li>
−
        </td>
+
            </ul>
−
         <td>Yes</td>
+
        </div>
−
      </tr>
+
        <div class="col-sm-3">
−
      <tr>
+
            <div class="four-e-box" style="background: rgb(170,179,0)">Environment &amp; Safety</div>
−
    <td>Polymerase Chain Reaction (PCR)</td>
+
            <ul>
−
    <td>
+
                <li>Can we reduce risks associated with using GMOs in healthcare technologies?</li>
−
    <ul>
+
                <li>How can we ensure our product is sustainable and environmentally friendly?</li>
−
    <li>Molecular detection of <em>T. cruzi</em> DNA is performed using a combination of three real-time PCR assays.</li>
+
                <li>How will the end-user dispose of any used materials?</li>
−
    <li>Acceptable specimen types are EDTA blood, heart biopsy tissue or cerebrospinal fluid.</li>
+
            </ul>
−
    </ul>
+
        </div>
−
    </td>
+
    </div>
−
    <td>
+
−
    <ul>
+
−
    <li>Allows high sensitivity in the acute phase</li>
+
−
    <li>Allows the presence of <em>T.cruzi</em> to be accurately distinguished from <em>T. rangeli</em></li>
+
−
    <li>Allows direct detection of infection and easy interpretation of results</li>
+
−
    </ul>
+
−
    </td>
+
−
    <td>
+
−
    <ul>
+
−
    <li>High variation in accuracy and lack of international quality controls</li>
+
−
    <li>High cost and complexity means it is not practical to use in a clinical practice</li>
+
−
    <li>Further validation is needed to prove whether PCR is suitable to diagnose the chronic phase of Chagas</li>
+
−
    </ul>
+
−
    </td>
+
−
    <td>Yes</td>
+
−
      </tr>
+
−
      <tr>
+
−
      <td>Serological tests</td>
+
−
      <td>
+
−
      <ul>
+
−
      <li>Detection of antibodies against <em>T. cruzi</em></li>
+
−
      <li>Includes techniques such as indirect fluorescent antibody (IFA) test, a commercial enzyme immunoassay (EIA) and immunochromatographic tests</li>
+
−
      </ul>
+
−
      </td>
+
−
      <td>
+
−
      <ul>
+
−
      <li>Can be used for acute phase and chronic phase</li>
+
−
      <li>High specificity and sensitivity</li>
+
−
      <li>Commercialised and approved for use by WHO</li>
+
−
      <li>Low-cost formats are available</li>
+
−
      </ul>
+
−
      </td>
+
−
      <td>
+
−
      <ul>
+
−
      <li>Cross reactivity can occur with diseases, such as leishmaniasis and schistosomiasis </li>
+
−
      <li>Performance of these tests is lower than reported by their manufacturer, especially against specific strains of <em>T. cruzi</em></li>
+
−
      <li>Not suitable for immunocompromised patients and newborns</li>
+
−
      </ul>
+
−
      </td>
+
−
      <td>No</td>
+
−
  </tr>
+
−
    </tbody>
+
−
  </table>
+
−
  <p>The table highlights the lack of a rapid and feasible diagnostic for congenital Chagas disease. Moreover, in June 2016 the WHO and experts on Chagas disease based in Latin America regarded a point-of-care diagnostic for congenital Chagas as their top priority in terms of the diagnostic needs for Chagas disease. The diagnostic needs were ranked following considerations of existing diagnostic tools and the expected clinical and epidemiological scenario of Chagas disease in the next five years. Strategies to tackle Chagas disease in an optimal fashion using our diagnostic device in the current Latin American society in Bolivia is outlined in our public policy proposal that can be found below: <a href="https://2017.igem.org/Team:Oxford/Chagas_Public_Policy"><img class="img-responsive" width="100px;" src="https://static.igem.org/mediawiki/2017/9/98/T--oxford--chagas_disease--button.png"></a></p>
+
−
  <h2>Treatment</h2>
+
−
  <p>An 8 week course of benznidazole or nifurtimox can be used to kill the parasite and treat Chagas disease. The younger the patient and the closer to acquisition of the infection, the higher the probability of parasitologic cure. Therefore, newborns with congenital Chagas disease have the greatest chance for cure, with data from Argentina indicating that the cure rate is higher than 90% if treatment is given within the first year of life. In most cases the potential benefits of medication in curing, preventing or delaying Chagas is balanced against the possible adverse reactions that occur in up to 40% of treated patients. However, newborns are least affected by side effects of  benznidazole or nifurtimox, due to the lower weight-accounted dosage, making treatment a very viable option. If the chronic phase is left untreated, additional specific treatment for cardiac or digestive manifestations may be required.</p>
+
−
  <h2>References</h2>
+
−
  <p>Louis V Kirchhoff, 2017. <em>Chagas Disease (American Trypanosomiasis) Treatment &<br/>
+
−
Management.</em> [Online] Available at<br/>
+
−
<a href="https://emedicine.medscape.com/article/214581-treatment"></a> [Accessed September 2017].</p>
+
−
<p>WHO, 2017. <em>Chagas disease (American trypanosomiasis) - Control strategy.</em> [Online]<br/>
+
−
Available at: <a href="http://www.who.int/chagas/strategy/en/"></a> [Accessed October 2017].</p>
+
−
<p>Ana María Cevallos and Roberto Hernández, 2014. <em>“Chagas’ Disease: Pregnancy and<br/>
+
−
Congenital Transmission,” </em>BioMed Research International, vol. 2014, Article ID 401864, 10<br/>
+
−
pages. doi:10.1155/2014/401864</p>
+
−
<p>Hemmige, V., Tanowitz, H., & Sethi, A., 2012. <em>Trypanosoma cruzi infection: a review with<br/>
+
−
emphasis on cutaneous manifestations. International Journal of Dermatology</em>, 51(5),<br/>
+
−
501–508. <a href="http://doi.org/10.1111/j.1365-4632.2011.05380"></a>.
+
−
<p>Marin-Neto, J. A., Cunha-Neto, E., Maciel, B. C., & Simões, M. V. 2007 <em>Pathogenesis of<br/>
+
−
chronic Chagas heart disease</em>. Circulation, 115(9), 1109-1123.
+
  
−
 
  
 +
    <h3>Cell-free Systems</h3>
 +
    <p>
 +
    One of the first things we realised when we first began to plan our kit was the current difficulties with using cells as part of our system; they would require a cold chain for transportation and maintenance of the culture once they had arrived in location and were being used for the kit. Whilst reading around synthetic biology in general we discovered a paper from Pardee at al. (2016), which described a method for freeze-drying cell lysate to then be used as a cell-free transcription/translation system. This was perfect, as there was no need for a cold chain, and the lysate could be produced cheaply and easily. At the open plant forum we received lots of ideas from talks by Jim Swartz and Keith Pardee, then we discussed our project with Keith Pardee. He gave us lots of practical advice on designing an optimal circuit for cell-free expression, and we decided to add pre-synthesised TetR to our kit rather than producing it in our kit, as we’d originally planned on doing.
 +
    </p>
 +
    <p>After further reading, including papers from Garamella et. al (2016), we further improved the theoretical design of our kit by discovering that we could use linear DNA from a PCR reaction for our kit, rather than plasmid DNA. This almost eliminates the risk of contaminating the environment, as bacteria will not take linear DNA up nearly as easily as plasmid.
 +
    </p>
 +
    <p>The final DNA-based part of the kit would be:</p>
 +
    <ul>
 +
        <li>Cell lysate, which can be mass-produced</li>
 +
        <li>PCR product ofr our circuitry thtat produces the TEV protease,</li>
 +
        <li>The TetR with the specific cleavage sequence that will be produced separately and added to the reaction.</li>
 +
    </ul>
 +
    <p>These would all be freeze-dried in the well of our kit.</p>
 +
    <p>
 +
    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/3/31/T--oxford--applied--design--fig3.jpg" />
 +
    </p>
 +
 +
    <h3>Initial Design Iterations</h3>
 +
    <p>
 +
    Our starting point involved the detection of cruzipain in a sample of blood, however we needed to design an output system and a method for reading the output. By applying our criteria to a cell-free diagnostic system, we were able to propose some early design options. We discussed these as a group, compared the relative merits of each, and decided which would be most suitable to carry on with.
 +
    </p>
 +
 +
    <h4>Early Stage: Output Ideas</h4>
 +
    <table class="table">
 +
        <tbody>
 +
            <thead>
 +
                <tr>
 +
                    <th></th>
 +
                    <th>Color Dye</th>
 +
                    <th>Clotting System</th>
 +
                </tr>
 +
            </thead>
 +
            <tr>
 +
                <th>Advantages</th>
 +
                <td>
 +
                    <ul>
 +
                        <li>Many current spot tests use colour as an indicator</li>
 +
                        <li>Clear presentation of the result</li>
 +
                    </ul>
 +
                </td>
 +
                <td>
 +
                    <ul>
 +
                        <li>Possible to design bacteria to produce a clotting inhibitor</li>
 +
                        <li>Can perform test on blood sample without requiring preparation</li>
 +
                    </ul>
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <th>Disadvantages</th>
 +
                <td>
 +
                    <ul>
 +
                        <li>Difficult to distinguish against the colour of blood</li>
 +
                        <li>Can't isolate plasma easily</li>
 +
                    </ul>
 +
                </td>
 +
                <td>
 +
                    <ul>
 +
                        <li>May be difficult to visualise clotting</li>
 +
                        <li>Result may be too subjective</li>
 +
                    </ul>
 +
                </td>
 +
            </tr>
 +
        </tbody>
 +
    </table>
 +
    <p></p>
 +
    <p>
 +
    From our research we found that the ‘clot-buster’ drug streptokinase is naturally produced by bacteria. This inspired us to use the properties of blood to our advantage; our output could interfere with the blood clotting system to produce a result.
 +
    </p>
 +
    <p>
 +
    With a coloured dye output, visualisation of the result would require the plasma to be isolated; this would increase the complexity and cost of the test.
 +
    </p>
 +
    <p>
 +
    Furthermore, a key goal for our applied design was to ensure it could be used in a scenario with minimal training and limited resources. Findings from the National Congenital Chagas Program in Bolivia (2004-2009) showed that follow-up after diagnosis was a major difficulty in controlling the disease; as such, an immediate bedside test would be most ideal. Isolation of plasma would require resources which may not be available at the bedside/in all healthcare settings.
 +
    </p>
 +
    <p>
 +
    Having reviewed these two options, we came to the conclusion that a system based around blood clotting as an output would be most suitable.
 +
    </p>
 +
 +
    <h4>Late Stage: Ideas to measure blood clotting output</h4>
 +
    <table class="table">
 +
        <tbody>
 +
            <thead>
 +
                <tr>
 +
                    <th></th>
 +
                    <th>Blood Collection Tube</th>
 +
                    <th>Microfluidics</th>
 +
                </tr>
 +
            </thead>
 +
            <tr>
 +
                <th>Advantages</th>
 +
                <td>
 +
                    <ul>
 +
                        <li>Equipment is easy to obtain</li>
 +
                        <li>Relatively cheap</li>
 +
                        <li>Would fit into current infrastructure</li>
 +
                    </ul>
 +
                </td>
 +
                <td>
 +
                    <ul>
 +
                        <li>Provides a quantitative measure of coagulation</li>
 +
                        <li>Decreases analysis time</li>
 +
                        <li>Can use a smaller volume of blood (fingerprick)</li>
 +
                    </ul>
 +
                </td>
 +
            </tr>
 +
        </tbody>
 +
    </table>
 +
    <p></p>
 +
    <ul>
 +
        <li>Blood Collection Tube: Our research showed that most blood collection tubes are lined with anticoagulation factors, in order to prevent blood from clotting. This inspired us to produce a collection tube lined with our freeze-dried cell-free system.</li>
 +
        <li>Microfluidics: Two papers published by Steckl et al. (Lab on a Chip 2014, Biomedical Microdevices 2017) inspired us to consider a new method to screen for blood coagulation. Steckkl and colleagues presented a cheap, point-of-care blood coagulation assay, which utilised microfluidics in a paper-based device.</li>
 +
    </ul>
 +
    <p></p>
 +
    <p>
 +
    Whilst simple, we decided that the blood collection tube method may not produce a clear result, which was an important consideration.
 +
    </p>
 +
 +
    <h3>Blood-clotting System: Hirudin vs Bivalirudin</h3>
 +
    <p>
 +
    Hirudin is a 65-amino acid peptide produced by leeches, and is used widely in the medical field as an anticoagulant. It is mass produced and purified using recombinant technology; initially hirudin was proposed as the output of our DNA/OMV systems, as recombinant hirudin expression in E. coli was shown to be efficient from the literature.
 +
    </p>
 +
    <p>
 +
    Applying our 4E’s framework to our decision to use hirudin led us to explore cost-friendly alternatives. We came across bivalirudin, a congener of hirudin with a similar mechanism of action. However, importantly, bivalirudin is a smaller peptide at only 20-amino acids in length. As a result, our cost analysis showed that it would cheaper to synthesise bivalirudin chemically than to produce hirudin recombinantly. This cost-difference provides a significant advantage in ensuring maximal availability of our kit.
 +
    </p>
 +
 +
    <h3>Integrating Out Ideas Into A Design</h3>
 +
    <p>
 +
    We began by sketching ideas on a whiteboard and sharing our thoughts during a group meeting.
 +
    </p>
 +
 +
    <h2>Current Kit</h2>
 +
    <h3> Cost </h3>
 +
    For costing of our kit we were aided by contact with David Sprent, an expert in International Supply Chain, and Juan Solano and Alfons Van Woerkom of the Global Fund. They helped us with costing, but also with the considerations that have to be taken into account when importing things into Latin America. We used Bolivia as a case study, and imagined what the situation would look like if the country were to adopt our kit wholesale, testing all 163,000 babies born every year. The kit would be manufactured in the UK and then transported to Bolivia, as according to <a href="https://2016.export.gov/industry/health/healthcareresourceguide/eg_main_092224.asp"> Export.gov </a> as of 2014 there was no local production of pharmaceuticals.
 +
    <h4> Materials </h4>
 +
   
 +
    <table class="table">
 +
        <tbody>
 +
          <thead>
 +
              <tr>
 +
                  <th>Component</th>
 +
                  <th>Cost Per Kit ($)</th>
 +
                  <th>Source </th>
 +
              </tr>
 +
          </thead>
 +
          <tr> <td> Bivalirudin </td> <td> 0.007 </td> <td> <a href="http://www.selleckchem.com/custom-peptide-synthesis.html">Cost (Selleckchem)</a> (Modelling told us we needed 50uM) </td></tr>
 +
          <tr> <td> Sodim Citrate </td> <td> 1.23^10-9 </td> <td><a href="https://www.researchgate.net/post/How_much_38_citrate_do_we_need_to_add_to_anticoagulate_blood_for_platelet_rich_plasma">Amount</a> </br>
 +
            <a href="http://www.sigmaaldrich.com/catalog/product/aldrich/w302600?lang=en&region=GB">Cost (Sigma Aldrich) </a>
 +
              </td></tr>
 +
          <tr> <td> Calcium </td> <td> 0.00311 </td> <td><a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1538-7836.2003.00075.x/full">Amount</a> </br>
 +
            <a href="http://www.sigmaaldrich.com/catalog/product/aldrich/449709?lang=en&region=GB">Cost (Sigma Aldrich)</a>
 +
              </td></tr>
 +
          <tr> <td> Tissue Factor </td> <td> 8.78*10^-9 </td> <td><a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1538-7836.2003.00075.x/full">Amount</a> </br>
 +
            <a href="http://www.abcam.com/recombinant-human-tissue-factor-protein-ab119148.html">Cost (abcam)</a>
 +
              </td></tr>
 +
          <tr> <td> Capillary Tube </td> <td> 0.074 </td> <td> <a href="http://www.sigmaaldrich.com/catalog/product/aldrich/z611182?lang=en&region=GB">Cost (Sigma Aldrich)</a></td></tr>
 +
          <tr> <td> Injection Molding of Kit </td> <td> 0.685 </td> <td> <a href="http://www.custompartnet.com/estimate/injection-molding/?units=1">Cost (CustomPartNet)</a></td></tr>
 +
          <tr> <td> Cardboard Box (70x30x50mm) </td> <td> 0.1 </td> <td> <a href="https://www.abcpackaging.co.uk/">ABCPackaging</a></td></tr>
 +
          <tr> <td> Microsafe Pipette </td> <td> 0.15 </td> <td> <a href="http://www.safe-tecinc.com/microsafe.htm">Cost (Safe-Tec)</a></td></tr>
 +
          <tr> <td> Printed Instructions </td> <td> 0.021 </td> <td> <a href="">?</a></td></tr>
 +
          <tr> <td> TetR </td> <td> 8.25*10^-5 </td> <td> <a href="https://www.mybiosource.com/prods/Recombinant-Protein/Tetracycline-repressor-protein-class-B-from-transposon-Tn10/tetR/datasheet.php?products_id=1056262">Cost (MyBioSource)</a> (Modelling told us we needed 100nM)</td></tr>
 +
          <tr> <td> Cell Lysate for DNA Reaction </td> <td> 0.9 </td> <td> <a href="http://www.cell.com/cell/fulltext/S0092-8674(16)31246-6?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867416312466%3Fshowall%3Dtrue">Pardee et al. (2016)</a></td></tr>
 +
          <tr> <td> <b> Total Cost </B> </td> <td> <b> 1.97 </b> </td></tr>
 +
        </tbody>
 +
    </table>
 +
 +
          <h4> Manufacturing Cost </h4>
 +
          <p> This was hard to estimate, given the unknowns in our kit, but we assumed we would ask a third-party to assemble the kit and this would lead to costs of around <b>$0.25</b> per/kit. </p>
 +
 +
          <h4> Transportation Costs </h4>
 +
          <p> We decided that with a minimum shelf life of around a year for our test it would be pertinent to send kits once a quarter to Bolivia, otherwise we risked them expiring before being used. With a 50x30x70mm box for our kit around 8,000 can fit on a europallet after taking into account further packaging for the pallet. This means we'd be sending 5 pallets/per quarter, and we estimated that this would cost around $12,000 per shipment to get from the factory in the UK to hospitals in Bolivia. This equates to around <b>$0.30</b> per kit. </p>
 +
 +
          <h4> Taxes </h4>
 +
          <p> Bolivia imposes a <a href"http://haiweb.org/wp-content/uploads/2015/08/Taxes-final-May2011a1.pdf"> <b>13%</b> </a> tax on pharmaceutical imports into the country.
 +
 +
          <h4> <b>Total Cost</b> </h4>
 +
            Totalling up all these costs, and then adding 25%, as was suggested to us by those we contacted, brings the total cost of our kit to around <b>$3.80</b>, which is significantly less than the current other options on the market.
 +
         
 +
       
 
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Revision as of 17:44, 30 October 2017

Applied Design

Introduction

iGEM encourages all teams to take their projects beyond the lab and to consider design using a holistic approach. The question “What is our real world problem?” has been a key consideration from the beginning and has guided our project throughout the summer.

To ensure we were putting our diagnostic device into context, we considered various aspects including safety, accessibility and socioeconomic factors in Latin America. Various design iterations were built upon over the course of the summer, influenced by discussions with experts from a range of disciplines - including blood coagulation, microfluidics and general diagnostic devices.

Having examined these design options more carefully, we propose a final design for our system which fulfills our criteria for a suitable diagnostic device.

Developing Our Design

Criteria for a suitable diagnostic device: considerations from the OpenPlant Forum

Our early design criteria was influenced by research into the challenges of designing new healthcare technologies in developing countries. Our research indicated that important considerations included the level of infrastructure present, the cost to the end-user and the amount of training required. (http://apps.who.int/iris/bitstream/10665/70543/1/WHO_HSS_EHT_DIM_10.13_eng.pdf)

In order to gain an insight into various aspects of synthetic biology, members of our team attended the OpenPlant Forum in Cambridge, UK. Dr Tempest van Schaik gave a talk titled ’Designing Diagnostics’, using her expertise in the development of bench-to-bedside healthcare technologies and importance of the end-user experience. From her talk, 3 key points stood out to us:

  1. Understand your analyte
    1. What is the context in which the kit will be understood?
    2. How exactly does taking blood work? Will we transport the blood to a different place, or do a spot-test by the bedside?
  2. Understand the users of the kit
    1. How will they be given the kit?
    2. Will they want to use it?
    3. Do they want it?
  3. Understand the diagnosis procedure
    1. What problems are encountered from an end-user perspective?
    2. Can we simulate a diagnostics procedure to discover an issues?

Having discussed these findings as a team, we proposed a set of general criteria to guide our initial design brainstorming.

Our 4E’s Applied Design Framework

Applied design is an important component of most iGEM projects, and requires an integrated and holistic approach to ensure projects are considered from a ‘real-world’ perspective. Based on our findings from OpenPlant and research, the Oxford iGEM 2017 team came up with a framework for considering applied design - the 4 E’s (‘Effectiveness’, ‘Ease of use’, ‘Economics’ and ‘Environment & Safety’).

This provides a structured method for applied design considerations, and we hope that this framework may prove useful for future iGEM teams.

Effectiveness
  • How long will it take to get a clear result?
  • How will we ensure the test it sensitive and specific?
  • How can we ensure it can be used at the bedside?
Ease of Use
  • What equipment would be necessary to use it?
  • How can we present the result clearly?
  • What level of training would be required for the end-user?
Economics
  • What materials will be used?
  • Will it be feasible to transport our device in large quantities?
  • How can we alter it to reduce the cost?
Environment & Safety
  • Can we reduce risks associated with using GMOs in healthcare technologies?
  • How can we ensure our product is sustainable and environmentally friendly?
  • How will the end-user dispose of any used materials?

Cell-free Systems

One of the first things we realised when we first began to plan our kit was the current difficulties with using cells as part of our system; they would require a cold chain for transportation and maintenance of the culture once they had arrived in location and were being used for the kit. Whilst reading around synthetic biology in general we discovered a paper from Pardee at al. (2016), which described a method for freeze-drying cell lysate to then be used as a cell-free transcription/translation system. This was perfect, as there was no need for a cold chain, and the lysate could be produced cheaply and easily. At the open plant forum we received lots of ideas from talks by Jim Swartz and Keith Pardee, then we discussed our project with Keith Pardee. He gave us lots of practical advice on designing an optimal circuit for cell-free expression, and we decided to add pre-synthesised TetR to our kit rather than producing it in our kit, as we’d originally planned on doing.

After further reading, including papers from Garamella et. al (2016), we further improved the theoretical design of our kit by discovering that we could use linear DNA from a PCR reaction for our kit, rather than plasmid DNA. This almost eliminates the risk of contaminating the environment, as bacteria will not take linear DNA up nearly as easily as plasmid.

The final DNA-based part of the kit would be:

  • Cell lysate, which can be mass-produced
  • PCR product ofr our circuitry thtat produces the TEV protease,
  • The TetR with the specific cleavage sequence that will be produced separately and added to the reaction.

These would all be freeze-dried in the well of our kit.

Initial Design Iterations

Our starting point involved the detection of cruzipain in a sample of blood, however we needed to design an output system and a method for reading the output. By applying our criteria to a cell-free diagnostic system, we were able to propose some early design options. We discussed these as a group, compared the relative merits of each, and decided which would be most suitable to carry on with.

Early Stage: Output Ideas

Color Dye Clotting System
Advantages
  • Many current spot tests use colour as an indicator
  • Clear presentation of the result
  • Possible to design bacteria to produce a clotting inhibitor
  • Can perform test on blood sample without requiring preparation
Disadvantages
  • Difficult to distinguish against the colour of blood
  • Can't isolate plasma easily
  • May be difficult to visualise clotting
  • Result may be too subjective

From our research we found that the ‘clot-buster’ drug streptokinase is naturally produced by bacteria. This inspired us to use the properties of blood to our advantage; our output could interfere with the blood clotting system to produce a result.

With a coloured dye output, visualisation of the result would require the plasma to be isolated; this would increase the complexity and cost of the test.

Furthermore, a key goal for our applied design was to ensure it could be used in a scenario with minimal training and limited resources. Findings from the National Congenital Chagas Program in Bolivia (2004-2009) showed that follow-up after diagnosis was a major difficulty in controlling the disease; as such, an immediate bedside test would be most ideal. Isolation of plasma would require resources which may not be available at the bedside/in all healthcare settings.

Having reviewed these two options, we came to the conclusion that a system based around blood clotting as an output would be most suitable.

Late Stage: Ideas to measure blood clotting output

Blood Collection Tube Microfluidics
Advantages
  • Equipment is easy to obtain
  • Relatively cheap
  • Would fit into current infrastructure
  • Provides a quantitative measure of coagulation
  • Decreases analysis time
  • Can use a smaller volume of blood (fingerprick)

  • Blood Collection Tube: Our research showed that most blood collection tubes are lined with anticoagulation factors, in order to prevent blood from clotting. This inspired us to produce a collection tube lined with our freeze-dried cell-free system.
  • Microfluidics: Two papers published by Steckl et al. (Lab on a Chip 2014, Biomedical Microdevices 2017) inspired us to consider a new method to screen for blood coagulation. Steckkl and colleagues presented a cheap, point-of-care blood coagulation assay, which utilised microfluidics in a paper-based device.

Whilst simple, we decided that the blood collection tube method may not produce a clear result, which was an important consideration.

Blood-clotting System: Hirudin vs Bivalirudin

Hirudin is a 65-amino acid peptide produced by leeches, and is used widely in the medical field as an anticoagulant. It is mass produced and purified using recombinant technology; initially hirudin was proposed as the output of our DNA/OMV systems, as recombinant hirudin expression in E. coli was shown to be efficient from the literature.

Applying our 4E’s framework to our decision to use hirudin led us to explore cost-friendly alternatives. We came across bivalirudin, a congener of hirudin with a similar mechanism of action. However, importantly, bivalirudin is a smaller peptide at only 20-amino acids in length. As a result, our cost analysis showed that it would cheaper to synthesise bivalirudin chemically than to produce hirudin recombinantly. This cost-difference provides a significant advantage in ensuring maximal availability of our kit.

Integrating Out Ideas Into A Design

We began by sketching ideas on a whiteboard and sharing our thoughts during a group meeting.

Current Kit

Cost

For costing of our kit we were aided by contact with David Sprent, an expert in International Supply Chain, and Juan Solano and Alfons Van Woerkom of the Global Fund. They helped us with costing, but also with the considerations that have to be taken into account when importing things into Latin America. We used Bolivia as a case study, and imagined what the situation would look like if the country were to adopt our kit wholesale, testing all 163,000 babies born every year. The kit would be manufactured in the UK and then transported to Bolivia, as according to Export.gov as of 2014 there was no local production of pharmaceuticals.

Materials

Component Cost Per Kit ($) Source
Bivalirudin 0.007 Cost (Selleckchem) (Modelling told us we needed 50uM)
Sodim Citrate 1.23^10-9 Amount
Cost (Sigma Aldrich)
Calcium 0.00311 Amount
Cost (Sigma Aldrich)
Tissue Factor 8.78*10^-9 Amount
Cost (abcam)
Capillary Tube 0.074 Cost (Sigma Aldrich)
Injection Molding of Kit 0.685 Cost (CustomPartNet)
Cardboard Box (70x30x50mm) 0.1 ABCPackaging
Microsafe Pipette 0.15 Cost (Safe-Tec)
Printed Instructions 0.021 ?
TetR 8.25*10^-5 Cost (MyBioSource) (Modelling told us we needed 100nM)
Cell Lysate for DNA Reaction 0.9 Pardee et al. (2016)
Total Cost 1.97

Manufacturing Cost

This was hard to estimate, given the unknowns in our kit, but we assumed we would ask a third-party to assemble the kit and this would lead to costs of around $0.25 per/kit.

Transportation Costs

We decided that with a minimum shelf life of around a year for our test it would be pertinent to send kits once a quarter to Bolivia, otherwise we risked them expiring before being used. With a 50x30x70mm box for our kit around 8,000 can fit on a europallet after taking into account further packaging for the pallet. This means we'd be sending 5 pallets/per quarter, and we estimated that this would cost around $12,000 per shipment to get from the factory in the UK to hospitals in Bolivia. This equates to around $0.30 per kit.

Taxes

Bolivia imposes a 13% tax on pharmaceutical imports into the country.

Total Cost

Totalling up all these costs, and then adding 25%, as was suggested to us by those we contacted, brings the total cost of our kit to around $3.80, which is significantly less than the current other options on the market.