Team:Austin LASA/Kit

h(g.Page, {title: 'Kit Considerations', prev: 'https://2018.igem.org/Team:Austin_LASA/Attributions', next: 'https://2018.igem.org/Team:Austin_LASA/Parts', selector: [2, 6]},
  h('p', null, 'Regarded by itself, any research study yields data that advances a particular field. The challenge thereby remains in successfully applying the research to its intended target group, a task that requires awareness and planning.  In order to better integrate our research to address HIV-1 detection in infants located in rural areas, we contacted various officials for experience and advice. Among these professionals include those with experience in conducting HIV research in the field, professionals who work with safety, and NGO officials associated with this disease. As a result of these correspondences, we were able to formulate appropriate plans and modified our project accordingly as mentioned below.'),
  h('p', null, 'Brief summaries of all communications will be included below, however, the key areas that were discussed have been detailed here. Amongst the various issues that were brought up, three main issues were commonly discussed: communication, cost, and sterility. Our team’s plan regarding all problems and associated modification of our project have been described as follows.'),
  h('p', null, 'In a rural setting, it is given that hospitals and labs have limited resources. As such, the abilities and restrictions of the patient must be properly considered, in order to alleviate stress on the hospital. For instance, many mothers travel to centrally located hospitals to receive care but return home within 24 hours to resume their normal life. Thereby, their time in the hospital is limited. Even though many people in these rural villages have phones, hospitals lack the manpower to communicate results and follow up with patients once they leave its vicinity. As a result, many people are not aware of their health conditions, even after being tested. As such, our proposed diagnostic kit has a quick turnaround rate in order to best suit the availability of the mothers. The protocols run in our detection kit allow the testing to be conducted within a 6 hour turnaround period. After results have been procured, they will be securely shared with the infant’s mother (in order to protect the patient’s privacy), and information regarding further treatment can be provided at this point.'),
  h('p', null, 'Additionally, when conducting any project, cost estimates are important as well. Many rapid detection tests for HIV are fairly reasonable, most of them being under $100. In an effort to keep costs low, our proposed test utilizes protocols that can be done in very resource limiting environments. For instance, many HIV detection tests utilize PCR which requires a thermocycler or similar environment that can replicate varying temperatures. With our planned protocol, a thermocycler is unnecessary and in its stead, a fixed temperature water bath can be used. Similarly, all reagents and enzymes are aliquoted to prevent contamination or spoiling or large quantities of materials. In addition, if this was a real venture, proper funding would be established whether it be from the government or another organization to support the implementation of this test.'),
  h('p', null, 'As with any detection protocol, sterility is crucial. A controlled workspace in central to any accurate testing, however, this environment is not feasible in many low resource facilities. In rural labs, not only does equipment tend to be lacking, but the training of the nurses and lab technicians is not on par with those in other countries. Since these individuals are the ones using the kit, it is important to provide adequate training and information to ensure proper use. In an attempt to address this issue, we have proposed to include safety and sterility protocols along with our kit. By providing additional information and a reference for standard aseptic technique, we hope to alleviate some of the discrepancies that may arise from different educational backgrounds. As previously mentioned, the design of our project encourages sterility. All reagents are lyophilized to increase resistance in harsh environments. Likewise, all enzymes and reagents are aliquoted to prevent cross-contamination. Moreover, in order to collect reliable results, all testing with the diagnostic kit will use both positive and negative controls throughout the entirety of the protocol to ensure contamination has been prevented at every stage.'),
  h('p', null, 'As a result of our interviews, we began drafting a sample training protocol recommended by Dr. Leautaud. You can access our sample training protocol here.'),
  h('p', null, 'We’ve included interview summaries below.'),
  h('br'),
  h(g.Section, {title: 'Dr. Veronica Leautaud, Ph. D.'},
    'Director of Education',
    h('br'),
    'Rice 360 Institute for Global Health',
    h('p', null, 'Dr. Leautaud is a professor at the Rice 360 Institute for Global Health and was able to provide the following feedback on our project and kit.'),
    h('ul', null,
      h('li', null, 'Discussion Regarding Blood Sample to LAMP Procedures',
        h('ul', null,
          h('li', null, 'Dr. Leautaud recommends that for the LAMP assay to start with white blood cells. If we begin with plasma, after lysing we will still want to have white blood cells present, but she adds that “The good thing about LAMP is that it is a robust amplification so you can still get away with lysing the WBC with a small amount of detergent or high temperature or some other mechanical approach without killing the LAMP enzymes. And without having to purify so much, normally [compared]  with PCR you need to clean it so much before the Taq polymerase works.”'),
          h('li', null, 'Overall the initial starting point would be to start with whole blood, with both red and white cells, then lyse the white blood cells (what we are after for the double-stranded DNA template)')
        )
      ),
      h('li', null, 'Blood Sample to Amplification Procedures/Advice',
        h('ul', null,
          h('li', null, 'Dr. Leautaud’s advice, “It is not trivial, but there are papers… There is one professor, David Kelso, he takes whole blood, takes a finger prick on a wet mount, flushes it with a bit of sodium hydroxide (diluted) which destroys the cells and even denatures the DNA a bit, but in the end results in clean DNA for easy sample prep.”'),
          h('li', null, 'Overall, the means of procuring a blood sample and processing it until it is usable in amplification studies has its own challenges. In terms of our team, it would be in our best interest to focus on one part of the planned kit. Generally, in a larger lab, different individuals would work on various parts of such a kit, so it is not realistic to plan on developing a fully functional kit (in theory) with such limited resources and time. This part is best suited for a collaboration with another lab.')
        )
      ),
      h('li', null, 'Dr. Leautaud’s Experience in Malawi',
        h('ul', null,
          h('li', null, 'Dr. Leautaud was developing a detection assay for HIV RNA in 2012 in which she focused on plasma (since it dealt with RNA).'),
          h('li', null, 'She describes the workspace as follows, “In Malawi, there is no lab. They had a separate room with a fridge, centrifuge, agar plates, did not have pipettes or a vortexer; Eppendorf tubes and gloves were scarce.”',
            h('ul', null,
              h('li', null, '“There are centralized locations that will look at PCR, made out of blood spots that will be taken when a baby is born, it is analyzed by a large microbiology lab with all the staff and materials needed. Two types of locations, local labs with no or minimal resources and large centralized national lab.”'),
              h('li', null, '“One major problem in Africa or Malawi is that there is no communication and follow up with patients and the hospital, so even if a child is diagnosed the parent will not know. The hospitals don’t have the manpower to do that.”')
            )
          ),
          h('li', null, 'She describes various parameters that should be considered, “The test needs to be under 3 or 4 hours for the mother to stay in the hospital.',
            h('ul', null,
              h('li', null, ' You also need to keep in mind cost.'),
              h('li', null, 'The third parameter is your user which would be the nurse or lab technician, who is not as highly trained as they are in America. Instead, they have technical degrees. You need to teach them to not contaminate the test. You need to be aware of the level of literacy and skill of the user. My test in Malawi was a fail, it was too sensitive and was always contaminated.'),
              h('li', null, 'Dr. Leautaud sent materials with visiting staff to Malawi, used lyophilized reagents.',
                h('ul', null,
                  h('li', null, 'She mentions considering that most products will be kept at room temperature, so to keep that in mind when designing the project.')
                )
              ),
              h('li', null, '“Be aware of the resources: whether you need electricity or will use battery powered machines?”',
                h('ul', null,
                  h('li', null, 'One research fellow that works with Dr. Leautaud mentions that she is planning on conducting a usability study to see if people in the area are willing to take time out of their schedule to learn how to use the technology/ device she created.'),
                  h('li', null, 'important to consider public interest and involvement')
                )
              )
            )
          )
        )
      ),
      h('li', null, 'Precautionary Measures to Prevent Contamination (Other than sending material directly)',
        h('ul', null,
          h('li', null, 'Dr. Leautaud mentions, “A good option is to aliquot your products or enzymes. You also need to make sure that when you are running your tests that you have a negative control, positive control, and sample.”'),
          h('li', null, 'When she conducted her tests, Dr. Leautaud used synthetic oligos as a positive control  and plasma as the negative control. She would follow through the entire process with the controls, not just add it in at a certain step to make sure there was no contamination of the data.')
        )
      ),
      h('li', null, 'Advice for Project Planning',
        h('ul', null,
          h('li', null, 'Dr. Leautaud recommended ordering LAMP immediately as it will take a while to arrive.'),
          h('li', null, 'She recommends to focus on the sensitivity of assays (explains method on how to do so).',
            h('ul', null,
              h('li', null, '“Start from the beginning, when you are receiving a blood sample from a baby you most likely will do a heel prick which is no more than 50 microliters on a good day, if you do a venous draw, no doctor will allow you to have more than 0.1 mL. So how many white blood cells will be present? Assume that there will be 10 HIV copies in that 50 microliters of the blood sample, then you must look at LAMP; you have 20 microliter LAMP reaction and 50 microliters, so you can identify how much the HIV copies are present. From there you can identify how many copies you will need for the CRISPR assay to work and how many for the fluoresce to work (find the limit of detection of the LAMP assay).”')
            )
          ),
          h('li', null, 'Dr. Leautaud recommends doing the experiment with the fluorophore specifically because even if it says that it is visible to the naked eye, it is important to identify the concentration that must be present for it to be visible?'),
          h('li', null, 'She recommends to focus on the CRISPR assay with a really clear limit of detection and characterize the assay (do a dilution series of the assay).'),
          h('li', null, 'Dr. Leautaud adds that we can consider a paper test instead of a test tube if that is better suited to our goal.'),
        )
      )
    )
  ),
  h(g.Section, {title: 'Jessie Anderson'},
    'Research Fellow',
    h('br'),
    'Rice 360 Institute for Global Health',
    h('p', null, 'Ms. Anderson visited Malawi as preparation for her own validation study but was able to provide valuable insight into the conditions of the hospital and staff in other parts of the world. She was able to provide insight into the functionality the neonatal wards at two tertiary, referral hospitals and was able to give us the following feedback.'),
    h('ul', null,
      h('li', null, 'Would you be able to describe the setting of the hospitals that you visited? What were the conditions? How many staff and how well trained were they?',
        h('ul', null,
          h('li', null, 'The neonatal wards for both QECH and KCH seemed understaffed, even for this season which is not the busiest of the year. There were two short (10-30 seconds long) power outages within 20 minutes in the KCH ward. KCH had more equipment available to them than QECH (much more incubators, radiant warmers, blue-lights, etc; I think also a higher proportion of the room heaters was working), but the flow and size of space available seemed better at QECH. Still not ideal at either location. Both wards were segmented off into low-risk, high-risk, and isolation/quarantine areas or rooms. Every 2 hours the mothers come for feeding at both sites, and once every morning nurses go on rounds to check up on every patient (a doctor accompanies at QECH; they only come if called at KCH).'),
          h('li', null, 'Overall the initial starting point would be to start with whole blood, with both red and white cells, then lyse the white blood cells (what we are after for the double-stranded DNA template)')
        )
      ),
      h('li', null, 'Blood Sample to Amplification Procedures/Advice',
        h('ul', null,
          h('li', null, 'Dr. Leautaud’s advice, “It is not trivial, but there are papers… There is one professor, David Kelso, he takes whole blood, takes a finger prick on a wet mount, flushes it with a bit of sodium hydroxide (diluted) which destroys the cells and even denatures the DNA a bit, but in the end results in clean DNA for easy sample prep.”'),
          h('li', null, 'As far as training, I am not able to know how well-trained the nurses are; every nurse in Malawi must go through a training provided by the COIN Manual, but I don’t know how often (if ever) it is repeated. For diagnosing of jaundice, talking with two different nurses at KCH yielded two different procedures in using the transcutaneous bilirubinometer (one answered us “I think everyone should be trained on it now..” and when asked how often it was calibrated “I have never seen it calibrated” – if it is supposed to be used the same as a very similar device, t is supposed to be calibrated every use).'),
          h('li', null, 'KCH is better staffed 24/7. At QECH, there are 3-4 nurses during the weekday, and 2 overnight and on weekends. The study nurse who was there told us 75% of deaths occur overnight.')
        )
      ),
      h('li', null, 'Are the hospitals equipped to handle diagnostic studies? How knowledgeable were the staff or patients on diagnostic testing/ how willing are they to get them done?',
        h('ul', null,
          h('li', null, 'Both of these central hospitals have partnered with various universities and organizations throughout the years to complete studies. Rice itself has primarily partnered with the neonatal and maternity wards of QECH over the past 10 years (starting with bubble CPAP), and other organizations (like Baylor and UNC-Chapel Hill) have done some work at KCH, though I’m not sure exactly what studies they’ve done. UNC has a permanent building at KCH, as does Rice and the Liverpool Welcome Trust at QECH. There is also currently a sepsis diagnostic study occurring at KCH as with another group funded by Greater Challenges Canada.'),
          h('li', null, 'The staff is knowledgeable at both locations of diagnostic tests; both QECH and KCH are very supportive of the BiliSpec jaundice-diagnostic study (we’ve met with the department heads of both locations as well as other clinicians). Also, because the grant that funds our study provides stipends for the nurses who work with us, they are willing to work beyond their normal work hours to have another source of income.'),
          h('li', null, 'Are there any problems with sanitation, resources, or communication that is present?',
            h('ul', null,
              h('li', null, 'The sanitation and resources at both central hospitals are not the same that’s present in the US (or most western) hospitals. That being said, nurses are supposed to use available hand sanitizer before and after each time they interact/touch a patient, and gloves and biohazard bins are used when drawing blood or performing heelpricks. As far as communication, the data management is not consistent or as thorough as in western hospitals. I think that is one of the biggest obstacles in performing studies in such an environment. For the BiliSpec study (and most studies should do this anyway), we will be providing all our material resources (i.e. devices, consumables like gloves and needles among other, computer, desk/chair, wifi dongle as wifi is not available, etc).')
            )
          )
        )
      )
    )
  ),
  h(g.Section, {title: 'Lindsey Dawson'},
    'Associate Director, HIV Policy',
    h('br'),
    'Kaiser Family Foundation',
    h('p', null, 'Ms. Dawson aided in understanding current HIV testing and was able to provide resources to help guide our own planned kit.'),
    h('ul', null,
      h('li', null, 'Ms. Dawson advised considering a person’s comfort level when using their DNA (saliva draw or blood draw).',
        h('ul', null,
          h('li', null, 'She stated that we should consider an individual’s perspective when deciding upon the testing we would use (heelprick vs blood draw)')
        )
      ),
      h('li', null, 'She recommended identifying why your test is better, as it will help with identifying how it will fare against others in the market',
        h('ul', null,
          h('li', null, 'Ms. Dawson mentioned that the CDC has FDA approved test online to use for market research if wanted.')
        )
      ),
      h('li', null, 'She mentioned considering the environment that your test can be used in.'),
      h('li', null, 'Ms. Dawson explained the importance of cost, in terms of the market, and states that we should aim at keeping the cost low.'),
      h('li', null, 'Ms. Dawson offered to send literature that is associated with existing kits',
        h('ul', null,
          h('li', null, 'Look at sample brochure that comes with HIV kits for a guide when designing the brochure/protocol tha  h(g.Section, {title: 'Dr. Veronica Leautaud, Ph. D.'},
    'Director of Education',
    h('br'),
    'Rice 360 Institute for Global Health',
    h('p', null, 'Dr. Leautaud is a professor at the Rice 360 Institute for Global Health and was able to provide the following feedback on our project and kit.'),
    h('ul', null,
      h('li', null, 'Discussion Regarding Blood Sample to LAMP Procedures',
        h('ul', null,
          h('li', null, 'Dr. Leautaud recommends that for the LAMP assay to start with white blood cells. If we begin with plasma, after lysing we will still want to have white blood cells present, but she adds that “The good thing about LAMP is that it is a robust amplification so you can still get away with lysing the WBC with a small amount of detergent or high temperature or some other mechanical approach without killing the LAMP enzymes. And without having to purify so much, normally [compared]  with PCR you need to clean it so much before the Taq polymerase works.”'),
          h('li', null, 'Overall the initial starting point would be to start with whole blood, with both red and white cells, then lyse the white blood cells (what we are after for the double-stranded DNA template)')
        )
      ),
      h('li', null, 'Blood Sample to Amplification Procedures/Advice',
        h('ul', null,
          h('li', null, 'Dr. Leautaud’s advice, “It is not trivial, but there are papers… There is one professor, David Kelso, he takes whole blood, takes a finger prick on a wet mount, flushes it with a bit of sodium hydroxide (diluted) which destroys the cells and even denatures the DNA a bit, but in the end results in clean DNA for easy sample prep.”'),
          h('li', null, 'Overall, the means of procuring a blood sample and processing it until it is usable in amplification studies has its own challenges. In terms of our team, it would be in our best interest to focus on one part of the planned kit. Generally, in a larger lab, different individuals would work on various parts of such a kit, so it is not realistic to plan on developing a fully functional kit (in theory) with such limited resources and time. This part is best suited for a collaboration with another lab.')
        )
      ),
      h('li', null, 'Dr. Leautaud’s Experience in Malawi',
        h('ul', null,
          h('li', null, 'Dr. Leautaud was developing a detection assay for HIV RNA in 2012 in which she focused on plasma (since it dealt with RNA).'),
          h('li', null, 'She describes the workspace as follows, “In Malawi, there is no lab. They had a separate room with a fridge, centrifuge, agar plates, did not have pipettes or a vortexer; Eppendorf tubes and gloves were scarce.”',
            h('ul', null,
              h('li', null, '“There are centralized locations that will look at PCR, made out of blood spots that will be taken when a baby is born, it is analyzed by a large microbiology lab with all the staff and materials needed. Two types of locations, local labs with no or minimal resources and large centralized national lab.”'),
              h('li', null, '“One major problem in Africa or Malawi is that there is no communication and follow up with patients and the hospital, so even if a child is diagnosed the parent will not know. The hospitals don’t have the manpower to do that.”')
            )
          ),
          h('li', null, 'She describes various parameters that should be considered, “The test needs to be under 3 or 4 hours for the mother to stay in the hospital.',
            h('ul', null,
              h('li', null, ' You also need to keep in mind cost.'),
              h('li', null, 'The third parameter is your user which would be the nurse or lab technician, who is not as highly trained as they are in America. Instead, they have technical degrees. You need to teach them to not contaminate the test. You need to be aware of the level of literacy and skill of the user. My test in Malawi was a fail, it was too sensitive and was always contaminated.'),
              h('li', null, 'Dr. Leautaud sent materials with visiting staff to Malawi, used lyophilized reagents.',
                h('ul', null,
                  h('li', null, 'She mentions considering that most products will be kept at room temperature, so to keep that in mind when designing the project.')
                )
              ),
              h('li', null, '“Be aware of the resources: whether you need electricity or will use battery powered machines?”',
                h('ul', null,
                  h('li', null, 'One research fellow that works with Dr. Leautaud mentions that she is planning on conducting a usability study to see if people in the area are willing to take time out of their schedule to learn how to use the technology/ device she created.'),
                  h('li', null, 'important to consider public interest and involvement')
                )
              )
            )
          )
        )
      ),
      h('li', null, 'Precautionary Measures to Prevent Contamination (Other than sending material directly)',
        h('ul', null,
          h('li', null, 'Dr. Leautaud mentions, “A good option is to aliquot your products or enzymes. You also need to make sure that when you are running your tests that you have a negative control, positive control, and sample.”'),
          h('li', null, 'When she conducted her tests, Dr. Leautaud used synthetic oligos as a positive control  and plasma as the negative control. She would follow through the entire process with the controls, not just add it in at a certain step to make sure there was no contamination of the data.')
        )
      ),
      h('li', null, 'Advice for Project Planning',
        h('ul', null,
          h('li', null, 'Dr. Leautaud recommended ordering LAMP immediately as it will take a while to arrive.'),
          h('li', null, 'She recommends to focus on the sensitivity of assays (explains method on how to do so).',
            h('ul', null,
              h('li', null, '“Start from the beginning, when you are receiving a blood sample from a baby you most likely will do a heel prick which is no more than 50 microliters on a good day, if you do a venous draw, no doctor will allow you to have more than 0.1 mL. So how many white blood cells will be present? Assume that there will be 10 HIV copies in that 50 microliters of the blood sample, then you must look at LAMP; you have 20 microliter LAMP reaction and 50 microliters, so you can identify how much the HIV copies are present. From there you can identify how many copies you will need for the CRISPR assay to work and how many for the fluoresce to work (find the limit of detection of the LAMP assay).”')
            )
          ),
          h('li', null, 'Dr. Leautaud recommends doing the experiment with the fluorophore specifically because even if it says that it is visible to the naked eye, it is important to identify the concentration that must be present for it to be visible?'),
          h('li', null, 'She recommends to focus on the CRISPR assay with a really clear limit of detection and characterize the assay (do a dilution series of the assay).'),
          h('li', null, 'Dr. Leautaud adds that we can consider a paper test instead of a test tube if that is better suited to our goal.'),
        )
      )
    )
  ),

h(g.Section, {title: 'Lindsey Dawson'},
    'Associate Director, HIV Policy',
    h('br'),
    'Kaiser Family Foundation',
    h('p', null, 'Ms. t will accompany our kit')
        )
      ),
      h('li', null, 'She details on how understanding the importance of detection is important, but how it is equally important to consider the steps that should be taken to get treatment afterward (eg. antiretroviral treatment)'),
      h('li', null, 'Sent the following information (even though the CDC material is focused on US primarily)',
        h('ul', null,
          h('li', null, h('a', {href: 'https://stacks.cdc.gov/view/cdc/23447'}, 'CDC recommendations on Lab testing for HIV')),
          h('li', null, h('a', {href: 'https://www.cdc.gov/hiv/pdf/testing/hiv-tests-laboratory-use.pdf'}, 'Lab tests')),
          h('li', null, h('a', {href: 'https://www.cdc.gov/hiv/pdf/testing/rapid-hiv-tests-non-clinical.pdf'}, 'Rapid tests')),
          h('li', null, h('a', {href: 'https://www.cdc.gov/hiv/pdf/testing/hiv-tests-advantages-disadvantages_1.pdf   '}, 'Advantages/Disadvantages to different HIV tests')),
          h('li', null, h('a', {href: 'https://stacks.cdc.gov/view/cdc/50872'}, 'Recommended testing algorithm')),
          h('li', null, h('a', {href: 'https://www.cdc.gov/hiv/testing/laboratorytests.html'}, 'More info here (incl. all the above links)')),
          h('li', null, h('a', {href: 'http://www.oraquick.com/What-is-OraQuick/OraQuick-In-Home-HIV-Test'}, 'OraSure’s home test')),
          h('li', null, h('a', {href: 'http://aidsinfo.unaids.org/'}, 'To see hardest hit countries, including where share of people unaware of their HIV status is highest (i.e. undiagnosed)'))
        )
      )
    )
  ),
  h(g.Section, {title: 'Christopher R Singh'},
    'Biological Safety Officer, MS, Ph.D.',
    h('br'),
    'The University of Texas at Austin',
    h('br'),
    'Environmental Health and Safety',
    h('p', null, 'Dr. Singh served as our guide for various safety concerns and was able to provide us information on various regulations as follows.'),
    h('ul', null,
      h('li', null, 'Based on the procedures and information provided, are there specific clearances or hazards that we need to be aware of?'),
      h('li', null, 'What is the best way to handle blood spots or DNA samples when using a detection kit? What are things that we should keep in mind for creating the most aseptic and safe kit?'),
      h('li', null, 'What are the best disposal or packaging methods when the goal is to use kits such as these overseas in non-lab environments?',
        h('ul', null,
          h('li', null, 'For the research side of things there would need to be approvals for working with human material, blood, cells etc. and working with HIV or the potential components, depending on the research there may be several groups that will look at the work including the IBC.  Research would need to be done in a BSL-2 space hopefully equipped with a biosafety cabinet.  The hazards with the research would of course be the human material as well as the virus or viral components. In the field there would be little opportunity for working aseptically unless the plan would be for a mobile lab to be in place. The best you can do there would be best practice or universal precautions, the idea being that you would treat all samples as potentially pathogenic or infectious, but there would likely not be an opportunity to set up a BSC or any type of clean work space.  You would want proper PPE which at a minimum would be clothes that cover skin (long sleeve etc.) gloves, eye protection and a lab coat.  Depending on how samples will be obtained there may be additional requirements needed.  All the waste should be hazardous material/biowaste, this means for the research side it will need to be autoclaved or disposed of in biohazard boxes.  For field work you would need to check country regulations since they often differ, but you would still want to consider it hazardous waste. Please let me know if I missed something or if anything needs clarification or if there is anything else I can help with.')
        )
      )
    )
  )
);