Difference between revisions of "Team:UiOslo Norway/Description"
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| − | This year’s iGEM team from UiO will address one of the increasing world problems and health treat, antibiotic resistance. For many years humans have evolved together with bacteria in an evolutionary race | + | This year’s iGEM team from UiO will address one of the increasing world problems and health treat, antibiotic resistance. For many years humans have evolved together with bacteria in an evolutionary race; for each new antibiotic produced, a simple bacteria will eventually be able to resist the medicine, survive the treatment and continue to grow. Today we have come to a place where there is little or no new antibiotics produced and the resistance of the ones that are in use is increasing. Without functional antibiotics modern medicine will fall back many centuries. Without antibiotics doctors won’t be able to perform basic surgeries, keep diabetes patients healthy, and treat pre-term babies from infections. It is postulated that antibiotic resistance may become a greater threat to our health than cancer, and by 2050, a frightening number of 10 million people is estimated to die due to infections caused by antibiotic resistant bacteria. |
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| − | Our goal is to make a diagnostic test associated with an app, which | + | Our goal is to make a fast, easy-to-use diagnostic test associated with an app, which would be available for medical personel both in the field and in the medical offices. In addition to this, we have made a descriptive scheme of a screening test that fits the mentioned criteria. |
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<p class="boxnotes leftext"> | <p class="boxnotes leftext"> | ||
| − | <b>Diagnostic test:</b> We will make a diagnostic test that will detect ESBL producing bacteria in urinary tract infections. The already established Penta Well test by Mura et. al. (2015), changes color from yellow to red when this resistant bacterium is present. The color change is due to the cleavage of the β-lactamring in Nitrocefin. The test can also discriminate between several classes of β-lactamases, and | + | <b>Diagnostic test:</b> We will make a diagnostic test that will detect ESBL producing bacteria in urinary tract infections. The already established Penta Well test by Mura et. al. (2015), changes color from yellow to red when this resistant bacterium is present. The color change is due to the cleavage of the β-lactamring in Nitrocefin. The test can also discriminate between several classes of β-lactamases, and serves as the perfect basis of our idea. |
</p> | </p> | ||
<p class="boxnotes leftext"> | <p class="boxnotes leftext"> | ||
| − | <b>Smartphone app:</b> The diagnostic test will be associated with a smartphone app – | + | <b>Smartphone app:</b> The diagnostic test will be associated with a smartphone app – PhoneLab. The camera on your smartphone will be connected to the app which by help of a binary code will detect any colorchange. Color will be cross-referenced with a database which will give the doctor a list of any resistant bacteria found and what antibiotics not to give. |
</p> | </p> | ||
<p class="boxnotes leftext"> | <p class="boxnotes leftext"> | ||
| − | <b>Screening test:</b> We want to use a simplified CRISPR/Cas9 system to link base-pairing to a detectable signal. The system will be comprised of a | + | <b>Screening test:</b> We want to use a simplified CRISPR/Cas9 system to link base-pairing to a detectable signal. The system will be comprised of a tracr/crRNA towards the dangerous resistance genes and a modified Cas9 protein. This will enable us to screen for the dangerous resistance genes, thus preventing antibiotics to end up in the wrong place even before the resistance gene products are already present. Conceptually; the tracr/crRNA is linked to a null-nuclease Cas9 that doesn´t cleave the target genes. Bound to the Cas9 is half of a detectable enzyme. By designing two, separate such constructs that recognize adjacent parts of the same gene, the two halves of a split-enzyme assay can be brought together and form a detectable construct. We imagine galactosidase as the effector in this split assay, which could detect for example X-gal derivative. Through realizing this, PhoneLab will become a functioning diagnostic tool on both a phenotypic and a genotypic level. |
</p> | </p> | ||
</div> | </div> | ||
Revision as of 10:32, 19 October 2016
Background
UiOslo team started to assemble in early February. By then we were 4 team members, and we started the long and time-consuming process of choosing an idea for our project. Medicine and health was themes that we were all concerned with, and several of the team members write master thesis in that area as well. We all agreed that public health is important and engaging. There were many good ideas, and we searched through the iGEM library to get inspiration for great ideas. From a molecular biologist perspective antibiotic resistance is learned early on in the bachelor degree at UiO. It is also something that is used frequently in research, and especially in synthetic biology, thus we started to think around the antibiotic resistance problem in the world.
Investigating todays problems with antibiotic resistance
After discussions with our supervisors and some research on our own we decided that our project should deal with antibiotic resistance. We wanted to get more insight of this problem and how a small group of 7 students could help solving or at least improve the antibiotic resistance situation in Norway and globally. Our supervisors got us in contact with the head of the microbiology department at Rikshospitalet (a part of the Oslo University Hospital) Tone Tønjum. She is also a professor and group leader of her own research group. The whole team met with her where she showed us around in her lab and we discussed the antibiotic resistance problem in Norway and how to address this problem.
Today’s detection methods
Tone made us aware of one particular problem. She talked about the diagnostic problems they had with antibiotic resistance and that the method they used today was not fast enough or too expensive for regular use.
A patient sample is grown on agar plates that may contain 12 different antibiotics zones. The plates need time to grow, usually 24h and then one can check for bacterial growth on the different antibiotic zones on the plate.
Rapid method exist also today Tone explained. But one huge limitation for most laboratories is high cost of these automated machines and maintenance. PCR may also be used for genotypic methods, such for the DNA sequence of interest. One drawback here is that the sequence or part of the sequence must be known, and even though a resistant gene is present there are differences in mode and level of expression which may make the treatment more problematic. Because of convenience, efficiency and cost the disk diffusion method is possibly the most widely used method in the world. Even though this is a convenient method to test for antibiotic resistance, it is time consuming and generates antibiotic waste. What Tone meant the marked and health institutions needs today is a fast, easy and cheap test to detect antibiotic resistance in every health institutions.
Extended spectrum beta lactamase
We discussed what kind of infections that had high prevalence and what kind of bacteria one should be concerned about. She talked about different resistant bacteria, and there was one type that particularly concerned Tone. This bacterium is producing extended spectrum β – lactamase. She talked about its development the last couple of years and its prevalence had not only increased in Norway but in the rest of the world as well the last several years. ESBL producing bacteria was something that the team had never heard about and we started doing a lot of research, and reading articles about this kind of bacteria.
We soon understood that we needed more expertise in this area and we contacted our team member Camilla’s previous professor Ole Andreas Løchen Økstad that works at the department of pharmaceutical biosciences. He has a lot of experience working with pathogenic bacteria and agreed to a meeting with us. He also seemed to think that this was an important project and very much needed. He gave us a lot of tips and also some of his contacts that were experts in resistant bacteria and ESBL.
Urinary tract infections
We also had to discuss what kind of sample we wanted to focus on. If it should be blood, urine, saliva etc. The team discussed this topic several times and eventually decided to focus on urine samples. There were many reasons for this, it’s easy to work with, the sample can be easily collected and it is relatively consistent for each sample taking as opposed to saliva where the sample depends on how much/little saliva/mucus you are able to cough.
We did a search in the prescription registry in Norway which is an online database where there is an overview of all prescribed medicine. Phenomethylpenicillin was the most prescribed antibiotic in Norway in 2014. Phenoxymethylpenicillin is a betalactam antibiotic and its actions is inhibited by ESBL producing bacteria. By deciding on urine as a sample we started also to investigate urinary tract infections and its occurrence both in Norway and in the rest of the world. All of this research was necessary to figure out if this is something the society could benefit from, and we wanted our project to be a part of a solution to the antibiotic resistance problem. Before we took any final decision we had to investigate whether or not a diagnostic test for urinary tract infections caused by ESBL producing bacteria could be beneficial for society. We went through a lot of research material and managed to get a good overview of urinary tract infections. UTI in general is the second most common infection caused by bacteria. Only airways infections are more common. UTI is usually generated from bacteria derived from the colon but other bacteria can also cause an infection. It affects both women and men, but women are more exposed to this type of infection due to physiological differences.
The diagnostic tool
We decided that UiOslo iGEM team should work on developing a diagnostic test for ESBL producing bacteria in urinary tract infections. Several experts in the field claimed that it’s necessary for the health sector today and such a test will contribute to reduce the use of antibiotic and contribute to more rightful use of antibiotic.
To set out for such a big task, we had a lot of work in front of us. One of the contacts Ole Andreas gave us lives in Tromsø in North of Norway and is a professor at the University of Tromsø and works at the National Center of Expertise on Antibiotic Resistance. He provided us with a lot of information on the ESBL situation in Norway, which they have researched for over a decade; he gave us a report where it clearly stated that ESBL prevalence in urinary tract infections had gradually increased over the years, especially from 2013 to 2014 where there had been an exponential increase (see graph below). He also provided us with different ESBL isolates they had collected from institutions from all over Norway. We wanted to use these isolates in our diagnostic test when it was finalized. By showing that our test works on clinical isolates will give the project a huge clinical relevance.
The UiOslo team is very excited about this year’s project and feels confident that the project may be beneficial to both patients and health institutions all over the world. The diagnostic test will be rapid, efficient and cheap, and it does not require any special training. The test is couplet to an app that will generate the results of the test. Because of this the team has utilized the multidisciplinary background that exists within the team and hopes that this year’s project has increased people’s knowledge and awareness of antibiotic resistant, and that UiOslo iGEM want to be a part of the solution of the increasing world problem: antibiotic resistance.
Description
This year’s iGEM team from UiO will address one of the increasing world problems and health treat, antibiotic resistance. For many years humans have evolved together with bacteria in an evolutionary race; for each new antibiotic produced, a simple bacteria will eventually be able to resist the medicine, survive the treatment and continue to grow. Today we have come to a place where there is little or no new antibiotics produced and the resistance of the ones that are in use is increasing. Without functional antibiotics modern medicine will fall back many centuries. Without antibiotics doctors won’t be able to perform basic surgeries, keep diabetes patients healthy, and treat pre-term babies from infections. It is postulated that antibiotic resistance may become a greater threat to our health than cancer, and by 2050, a frightening number of 10 million people is estimated to die due to infections caused by antibiotic resistant bacteria.
Today the second most common infection caused by bacteria is urinary tract infections, and the trend of this infections being caused by resistant bacteria has increased in the last years. ESBL-producing E. coli produce hydrolytic enzymes, β-lactamases, which have the ability to cleave and thereby inactivate antibiotics.
Our goal is to make a fast, easy-to-use diagnostic test associated with an app, which would be available for medical personel both in the field and in the medical offices. In addition to this, we have made a descriptive scheme of a screening test that fits the mentioned criteria.
Diagnostic test: We will make a diagnostic test that will detect ESBL producing bacteria in urinary tract infections. The already established Penta Well test by Mura et. al. (2015), changes color from yellow to red when this resistant bacterium is present. The color change is due to the cleavage of the β-lactamring in Nitrocefin. The test can also discriminate between several classes of β-lactamases, and serves as the perfect basis of our idea.
Smartphone app: The diagnostic test will be associated with a smartphone app – PhoneLab. The camera on your smartphone will be connected to the app which by help of a binary code will detect any colorchange. Color will be cross-referenced with a database which will give the doctor a list of any resistant bacteria found and what antibiotics not to give.
Screening test: We want to use a simplified CRISPR/Cas9 system to link base-pairing to a detectable signal. The system will be comprised of a tracr/crRNA towards the dangerous resistance genes and a modified Cas9 protein. This will enable us to screen for the dangerous resistance genes, thus preventing antibiotics to end up in the wrong place even before the resistance gene products are already present. Conceptually; the tracr/crRNA is linked to a null-nuclease Cas9 that doesn´t cleave the target genes. Bound to the Cas9 is half of a detectable enzyme. By designing two, separate such constructs that recognize adjacent parts of the same gene, the two halves of a split-enzyme assay can be brought together and form a detectable construct. We imagine galactosidase as the effector in this split assay, which could detect for example X-gal derivative. Through realizing this, PhoneLab will become a functioning diagnostic tool on both a phenotypic and a genotypic level.
Urinary Tract Infection
This text will contain facts about drugs and treatments that have a base in Norway. Different countries often use different drugs and treatments.
Cystitis, urinary tract infection and painful bladder disease are all names for the same disease. About 80-90% of the time, the infection is caused by the bacteria Escherichia Coli (E. Coli) - a gram-negative bacteria that lives in our intestines. When E. Coli (or another bacteria) makes its way through the ureteral and to the urinary bladder, we have an urinary tract infection. Urinary tract infection can be divided into two categories; upper urinary tract infection (which include infection of the kidneys, also called pyelitis), and lower urinary tract infection (which only infect the ureteral and the bladder). You may also call it uncomplicated urinary tract infection or complicated urinary tract infection.
The infection affects more women than men. It is a 100 times more common for women to get cystitis, than it is for men. The reason for that is biology and anatomy. Women have a shorter ureteral and therefore it is easier for the bacteria to infect the bladder. Also, after menopause, women often experiences that the mucosa gets dry. This makes it easier for the bacteria to infect organs.
So, how do we treat cystitis?
In Norway we have national guidelines that doctors use in the treatment of different kinds of infection. The guidelines are made to keep the total amount of antibiotics down, and to give the best and most effective treatment to the patient. It is based on the principle that we must use narrow-spectrum antibiotics before broad-spectrum antibiotics. This is to keep the antibiotic resistance at a distance.
Uncomplicated urinary tract infections and complicated urinary tract infections are treated differently. The body can often take care of an uncomplicated cystitis by itself, but many use antibiotics to shorten the duration of the disease.
As many as 40% of the E. Coli found in urinary tract infections in Norway have been seen to be resistant against amino penicillin, such as amoxicillin. The other amino penicillin have shown to be intermediate sensitive, and therefore amino penicillin are no longer first hand treatment against cystitis.
Other penicillin such as mecillinam penicillin can still be used, as about 91% of E. Coli are still sensitive. Less than 2% are resistant.
Trimethoprim, along with pivmecillinam and nitrofurantoin are considered as first hand treatment against lower urinary tract infections. Usually there is no need to treat the patient for more than three days. According to the national guidelines for antibiotics in Norway the first hand treatment looks like this:
- Trimethoprim: 160 mg 2 times a day or 300 mg in the evening for 1-3 days
- Nitrofurantoin: 50 mg 3 times a day for 3 days
- Pivmecillinam: 200 mg 3 times a day for 3 days
All these treatments are considered as equally adequate treatment. Note that this guide is considered for adults who are not pregnant.
A new study also suggests that NSAIDs can be used in the treatment of lower urinary tract infection. It is considered to be equally as good as quinolones.
Treatment against pyelitis is the same as for uncomplicated cystitis, but the treatment is of longer duration.
- Trimethoprim: 160 mg 2 times a day or 300 mg in the evening for 5-7 days
- Nitrofurantoin: 50 mg 3 times a day for 5-7 days
- Pivmecillinam: 200 mg 3 times a day for 5-7 days
Note that treatment depends on severity and response of treatment. There are other guidelines for pregnant women and children.
The doctors also have to consider many factors when deciding which treatment is the most adequate for the patient; such as kidney function, allergies, pregnancy (which trimester?), breastfeeding, age and so on.
PhoneLab
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Software
APP description
The app developed for use with the PhoneLab was devised with the thought of measuring the color change of the urine samples by using the diode in our camera as the photoreceiver. Since the color change might be minute and only small amount of bacteria are antibiotic resistant we want to be able to register these small amounts of color change in the samples.
At first we considered a full spectrophotometer but decided that just taking a picture under the right lighting circumstances would be adequate to measure color changes quite accurately.
So the IT department designed the app based on these criteria and chose to develop the app with Android SDK using Java's API to handle the pixel readout and baseline comparisons that would have to be made to account for the different nuances of urine.
More precisely explained the app has to account for the base sample of urine and store the unique RGB value of the urine sample without any reagents, so that it can be compared to the samples with reagent. After this comparison is made the color change from the pixel’s RGB values are evaluated as either significant enough color change to determine antibiotic resistance or not significant enough color change to determine antibiotic resistance.
The reason for using a camera diode instead of just looking with the naked eye is because of the sensitivity of the camera combined with controlled light environment should equate to more reliable measurements.
APP design
The app was created in Android SDK and AIDE (Android IDE) it features very simplistic design with some information about antibiotic resistance to help doctors choose suitable antibiotic alternatives in the case of a sample containing antibiotic resistant bacteria towards conventional antibiotics.
The application takes a picture into the phoneLab device and runs a quick test on the resulting bitmap from the picture taken.
The points of reference are the 7 dots on the example picture above, where the bottom sample has no reagent added to the urine so that the bottom sample is the color change reference point. The color change is then measured from this baseline RGB value and compared with the 6 other samples to evaluate if there has been a significant color change in any of them.
Hardware
It all started with our iGEM team wanting to build a diagnostic device to deal with the problem of antibiotic misuse. After deciding we were going to work with beta lactamase enzymes in urinary tract infections, we started with our first idea; detection of changes in pH caused by hydrolysis of the beta lactam ring found in all beta-lactam antibiotics. To do this, we initially imagined to design and use an ISFET device (Ion Sensitive Field Effect Transistor), but due to poor readouts with our lab equipment we decided after a while to move away from the pH-measurements and go for an optical solution.
Figure 1. The first sketches of Prothenius - an ISFET device run by an arduino computer along with a simple display, to detect the hydrolysis of beta lactam ring.
The optimal candidate for optical detection of beta lactamase proved to be nitrocefin, a molecule with a beta-lactam ring that does not function as an antibiotic. When beta lactamase hydrolyse its beta-lactam ring, nitrocefin turns red. This enabled us to do an optical measurement of the color change to determine the presence of bacteria resistant to beta-lactam antibiotics.
The first idea to tackle this problem of measuring colorchange was to build a spectrometer using Arduino computer and 3D printing, but due to casts, this would have been less viable to use in for example third world countries.
We therefore decided to utilize the power that recides in the mobile devices many carry around every day; the smartphone. The modern smartphones generally have very good cameras, powerful processor calculations, and it is easy to develop apps for them. To do so, we needed to design and make hardware that allowed us to interface our samples with a mobile camera, and further design software (an app) to work with this hardware.
Figure 2. The first sketches of Phonelab. The left sketch is the very first version which was changed to the middle sketch due to some optics problems. The sketch to the right is a single cuvette test.
The purpose of this design is to create a stable way to interface samples with the app. The app collects all the information needed to read and analyse the color distribution between cuvettes, based on the contents of each tube. It then compares the results to a pre-made list containing information on what we are looking for. It would be convinient to have a login-option with an interface where you can add projects and patients, with the possiblilty to add notes. It could also be possible to allow doctors to send prescriptions for the suitable medicine. This could, for example, be done with a QR code on sms. For more information about the app, see the “Software”-section.
Figure 3. The first drafts of the app that would analyze the colors that phonelab detects.
As in any project, there were several prototypes before arriving at our final design. The assembly of the first prototype of PhoneLab was used to find design flaws and proving that the project was possible. The problems we encountered were the focusing on the cuvettes in such a way that we get the most accurate color readout, and the color difference of each diode. We decided that the next model would feature a different geometry to fix these issues.
Figure 4. The assembly of the electronics for PhoneLab prototype 1.
The dimensions and shape of the PhoneLab Optics design with 5 tubes for the nitrocefin penta well test are shown below.This design is made with an exchangeable phone adapter to fit with many different phones. A small LED is included in each slot, to ensure a homogenous and stable illumination of the samples. The LEDs run of an internally installed battery pack, and the light intensity is adjustable. As you see there is equal distance to every slot, and light from the LEDs are emitted radially through the slots where the sample is placed, towards the origin where the lence is placed.
Figure 5. The geometry of PhoneLab Optics, made for perfect 3D prints and cuvette exposure to camera.
To further improve on our device, we imagine to coat it with a hydrophobic layer that repels polar molecules, thereby ensuring it stays clean when being used. We also want this device to autoclavable without being destroyed. In this way it can be easily sterilized, when using it on bacterial samples, and it can be reused in a safe manner. The current material is PVA plastic, meaning that we would have to change the material to make it autoclavable. The cool part about this product, in addition to the box itself, is that it interfaces of biological samples to a phone and gives the userbase the ability to store, geotag, and share information in such an easy way through our software.
To summarize; through a series of prototypes, we have designed and 3D-printed a device that interfaces urine samples, or any sample one has software to and interest in analyzing, with our PhoneLab.
Design
Design here
Contact
