|
|
Line 166: |
Line 166: |
| ) | | ) |
| ) | | ) |
− | )
| |
− | )
| |
− | )
| |
− | ),
| |
− | 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.'),
| |
| ) | | ) |
| ) | | ) |