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We are two 6th grade students from CSG Penta College Jacob van Liesveldt in Hellevoetsluis, The Netherlands. We want to contribute to global health improvement; particularly the health in third world countries which don't have the money themselves, though they do need the help. We were introduced to the Imagine project by our chemistry teacher, P. Dijkwel. He knew about our interest in DNA and diseases and suggested the project DNA-analyse of the Imagine School Competition. Lotte van de Berg made it possible for us to participate in the DNA-Analyse project. In this project we wanted to combine our interest in DNA and the possibility to contribute to the health of third world people.
Worldwide, infectious diseases are responsible for one third of the total amount of annual deaths. However, almost all of these deaths occur in third world countries because of the large prevalence of many health issues like malnutrition, lack of clean drinking water, poor hygiene and few medical resources. The HIV-virus is the prominent cause of death, claiming over 1.2 million lives in sub-Saharan Africa only. After HIV/AIDS and malaria comes tuberculosis in the list of top causes of deaths in third world countries. Tuberculosis is responsible for a total of 1.4 million deaths per year, with 95% of the deaths occurring in third world countries.
Because of these shocking statistics, we decided to base our project on the third world problem tuberculosis. Our project comprises a durable test to demonstrate whether a person has tuberculosis or not, and a description of the actual realisation of the project. The test, a combination of two already existing microbiological and molecular methods, has to be very simple to execute, cheap and it must produce reliable results. This is essential in order to make the test durable and attractive for practical use.
If properly applied, this test can largely reduce the number of tuberculosis deaths. Timely diagnosing increases the chance of effective antibiotic treatment and it reduces the risk on outbreaks. Being a relatively infectious and difficult disease to cure, timely diagnosing of tuberculosis is vital. That's why we want to be helpful at this point of the process, instead of treatment and cure.
Swaziland is a small country in the south of Africa with remarkably high death rates due to tuberculosis. Being a fairly unknown country, we thought that the best way to make a difference in this world would be to focus our efforts on this region; preferably testing all inhabitants, but starting with symptomatic patients and patients with other risk factors.
Table of contents
Disease & Cause 2
Risks & Tests 3
TB Test 4
Advantages & Disadvantages 6
''If the importance of a disease for mankind is measured by the number of fatalities it causes, then tuberculosis must be considered much more important than those most feared infectious diseases, plague, cholera and the like.''
- Robert Koch, discover of tuberculosis, father of bacteriology
In 1882 Robert Koch, a German physician and scientist, discovered the real cause of a terrifying disease called consumption. This disease had gained its name because it seemed that infected people were consumed from within, with a bloody cough, fever and long relentless wasting.
Nowadays due to high advance of science, we know a lot more about this global disease and we've got much better resources (antibiotics) to treat infected people. As we got to know more about the pathogenesis of this bacterial infection, we started calling it tuberculosis.
Robert Koch had demonstrated that the cause of tuberculosis lies within the tuberculosis bacilli, the scientific name being Mycobacterium tuberculosis (MTB). This micro-organism of only a few microns in length is capable of causing major damage to body organs.
Tuberculosis is transmitted primarily by breathing in microscopic airborne droplets containing the MTB. These tiny droplets are produced when infected individuals talk, cough, sneeze or spit, making tuberculosis a highly infectious disease.
FIGURE 1 Mycobacterium tuberculosis.
Pathogenesis - latent TB vs. active TB
If a person inhales infected 'air', the bacteria force their way down and spread through the respiratory tract. During this first stage of infection, which generally lasts for several months, the body's immune system can prevent the disease from spreading to other parts of the body. It does this by encapsulating the bacteria. The formation of tubercles, the signature of tuberculosis is a fact. MTB can't multiply within these tubercles because of the low pH. However, MTB can survive within tubercles for extended periods of time. During this time, the individual is asymptomatic and can't infect other people. This is known as the latent (or inactive) phase. In many cases, the disease doesn't develop beyond this stage.
Nevertheless, the infection can progress to the next stage: the active stage. This happens when the immune system is weakened and fails to stop or isolate the infection. That's why the co-infection with HIV/AIDS is such a deathly combination. In the active stage, the germ multiplies rapidly due to suitable conditions and destroys the tissues of the lungs. Now the individual shows symptoms and is able to infect others. But MTB doesn't only affect the lungs. During low immunity, MTB can escape into the bloodstream and be carried elsewhere in the body, destroying other body organs.
Tuberculosis is primarily transmitted by breathing in infected 'air'. This makes the lungs particularly vulnerable to this infectious disease because the lungs provide our body with oxygen from the air. Therefore frequent symptoms of active (pulmonary) tuberculosis are:
Deep and/or bloody coughing
Diminished respiratory capacity
When the disease spreads to other parts of the body through the lymph nodes and blood stream, other symptoms may occur. MTB mostly ends up in the kidneys, spinal cord and brains, leaving permanent, disabling scar tissue with all its consequences.
Tuberculosis must not be confused with other diseases. Many pulmonary diseases exhibit similar symptoms, like deep coughing, fever and weight loss. Some of these other conditions include:
Bacterial lung abscess (empyema)
Chronic obstructive pulmonary disease (COPD)
Infection with fungus (e.g. histoplasmosis)
Infection with other mycobacterium
However, this doesn't make testing on tuberculosis less worthwhile. Almost all of the diseases above require different treatments. Hence, it's very important to be able to distinguish between them for appropriate treatment.
Existing test methods
At the moment, there are not many effective methods of diagnosing tuberculosis. One of the most common used tests is the TB skin test. This method involves an injection of cell-free purified protein derivative (PPD) made from inactivated M. tuberculosis cultures. If a person has been previously infected with TB, the injection causes a skin reaction. Unfortunately, recent studies have shown that other things cause the skin reaction, too, resulting in false-positive outcomes.
Another frequently used test is the TB blood test which measures how the immune system reacts to the Mycobacteria tuberculosis. However, the World Health Organisation warns for misdiagnoses due to the inefficiency of these blood tests. The search for specific antigens and antibodies is very difficult. The chance on false-positive or false-negative results is high, because many antigens and antibody responses look very similar.
Two other tests include sputum smear microscopy and chest x-rays. Sputum smear microscopy is cheap and fast but low in sensitivity. Chest x-rays are on the other hand very expensive and only detect the presence of tubercles.
The risk for taking up latent TB is very high. A person needs to inhale only a few of these germs to become infected. It's estimated that one third of the world's population has latent TB. Possible risk factors for latent TB to develop into active TB are:
People with weakened immune systems, like HIV/AIDS-patients
People who became infected with TB bacteria in the last 2 years
Babies and young children, due to weak immune systems
People who use tobacco and/or drugs
People who were not treated correctly for TB in the past
The TB test is designed with the purpose to reduce tuberculosis-linked deaths in third world countries by timely diagnosing. In order to achieve this aim, the TB test must require the following:
It must be capable of producing reliable results
It must be attractive for practical use
It must be as sustainable as possible
Before going deeper into the test requirements, the principle of our test will be explained.
The TB test is a combination of two already existing methods:
The LAMP-method replaces PCR and is used for amplification of the target DNA. End-point fluorescence follows immediately, ensuring the observable detection of the target gene.
LAMP stands for Loop-mediated isothermal amplification. LAMP uses six different primers which are specifically designed to recognize six distinct regions on the target gene. Amplification (and detection) of a target gene can be completed in a single step, by incubating the mixture of sample, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature around 64Â°C. Unlike PCR, the LAMP method is an isothermal process and therefore requires no very high and alternating temperatures for denaturation of double stranded DNA. Instead, a special kind of DNA polymerase exhibits strand-displacement activity.
To amplify the DNA, the double strand of DNA has to be separated. This will be ensured by enzymes and artificially designed primers. Primers are starters, complementary to a specific part of the DNA, which grow to a DNA strand and separate it from the other strand. A couple of different primers are needed:
Primers functioning as 'duplicators'
Primers functioning as 'separators'
Primers functioning as 'loop-formers'
In the first part of the process, primers are used to form the dumbbell shaped structures (1), which serve as the starting structures for the amplification cycle. The amplification cycle (2) is where the multiplication actually starts. With the loop-mediated structures playing a key role, various sized structures consisting of repeated target sequences are formed exponentially in a relatively short time. LAMP is so efficient that DNA is being amplified 109 - 1010 times in 15-60 minutes.
Because of LAMP's high specificity, the target gene sequence can easily be detected just by judging presence of amplified products. The presence of amplified products can be indicated by end-point fluorescence.
After a LAMP reaction, large amounts of pyrophosphate ion by-product have formed. These ions react with magnesium ions (Mg2+) to form the insoluble product magnesium pyrophosphate. Calcein, a fluorescent chelating agent, initially chelates manganese ions. In other words, calcein forms a chemical complex in which the manganese ions are enclosed which causes the calcein to remain quenched. During the amplification, the generated pyrophosphate ions will progressively take away the manganese ions from the calcein molecules. This results in an emission of green fluorescence of the calcein when irradiated under natural, ultra violet or blue light.
Under ultra violet light
Under natural light
FIGURE 4 Observable detection of target gene
Getting back to test requirements, a proper test should be:
Productive and effective
A detection method must be productive and effective. It must be capable of producing reliable results in a relatively short time.
A productive and effective test is obviously attractive for practical use. To make the product more attractive, the test must be affordable and very easy to execute. Just a few simple actions with cheap equipment should lead to valid outcomes, making it ideal for detection in rural areas where proper resources and laboratories are scarce.
Does the TB test meet the requirements?
The test meets many of the requirements stated above. It is considered to be productive and effective, attractive for practical use and fairly sustainable. LAMP in combination with end-point fluorescence is known for its sensitivity and reliable outcomes. Some expensive equipment is required, though they have to be purchased only once. The costs per test are very low, which makes the test attractive for actual application.
The test's advantages and disadvantages:
Very sensitive and specific
Well-suited for limited resource situations
Difficult primer design
Small 'testing menu' (not suited for unknown or unsequenced targets)
Fairly expensive equipment
GeneralFile:Flag of Swaziland.svg
Swaziland is a very small, landlocked country in the south of Africa, bordered to the north, south and west by South Africa, and to the east by Mozambique. In Swazi, Swaziland's official language, the Kingdom of Swaziland is called Umbuso WeSwatini, named after the 19th century king Mswati II. English is the second spoken language.
FIGURE 2 Flag of Swaziland
Geography and climate
Swaziland has a surface area of 17.363 kmÂ², making it the smallest country on the southern hemisphere. The terrain of Swaziland can be roughly divided into mountainous in the West to descending to low field regions in the East. The highest point is the mountain the Emlembe, which is 1862 metres high. Swaziland has little natural resources in comparison to other African countries. There are only a few small gold, diamond, and coal deposits. Approximately 10,25% of the land is used for arable purposes. The rest of the land is either nature or urban area.
The climate is tropical to near temperate in the west. With temperatures reaching 40 Â°C and rainfall only occurring in summer, the country has to cope with droughts which enlargers the issue of limited supplies of potable water. Other environmental issues are soil degradation and erosion, overgrazing and decimated wildlife populations because of hunting.
People and health
Swaziland has a population of 1,386,914 people and a population density of 61 inhabitants per square mile. However, the western region is much more densely populated because in the west are more and larger cities. Most of the population has a job in agriculture, although about 40% is unemployed. Swaziland is mostly dependent on the import and export with South-Africa. The main export earner is sugar, together with wood pulp, but since the producer closed a couple of years ago there is only the sugar industry left.
Nearly 70% of the population lives to standards beneath the limit of poverty. Because of this great poverty and disastrous HIV-epidemics, half of the population doesn't reach the age of 60. Nowhere in the world are so many people infected with HIV of other infectious diseases like tuberculosis. It causes Swaziland to become socially and economically weaker, even to the point where the long-term survival of the whole country is at stake.
Despite of wide-spread modernisation, a lot of Swazi people have preserved their age-old culture and traditional ceremonies. The two most important ceremonies are the Incwala in December and the Umhlanga in late August or early September. The most supported religion in Swaziland is Zionism, which is a mixture of Christianity and indigenous ancestral worship. There are also a lot of Roman Catholics and Muslims.
In 2006 came a constitution in effect after the long monarchy of King Mswati III. He was pressured in the 1990s to allow political reform and democracy. However, later on he came back later on his promises. The legal status of political parties in Swaziland remains unclear. In 1968 independence was granted from the UK. The official Capital of Swaziland is Lobamba but Mbabane is the governmental Capital. Swaziland is divided in four districts in which it is governed: Hhohho, Lubombo, Manzini and Shiselweni.
In order to diagnose on tuberculosis, you first need to collect a sample from a possibly infected individual. Tuberculosis is mainly a pulmonary disease, so the best place to get samples from is the respiratory tract. Taking buccal cell-samples is a possibility, but sputum from the lungs is more preferable due to a higher chance on the presence of tuberculosis bacilli.
When MTB is collected, the DNA within has to be extracted from the cells. The DNA is obtained through a 4-step routine procedure:
Breaking the cells open (cell lysis) to expose the DNA within. This is done by chemical and physical methods of blending, grinding or sonicating the sample.
Removing membrane lipids by adding a detergent or surfactants.
Removing proteins by adding a protease.
Precipitating the DNA with an alcohol (usually ethanol or isopropanol). Since DNA is insoluble in these alcohols, it will clump together.
When the DNA freely floats in the solution, the LAMP reaction is put in action. LAMP is responsible for the amplification of the target DNA. Briefly explained, the following actions have to be executed:
Prepare the master mix, which contains: six primers, BST DNA polymerase, substrates, dNTP's, buffers and calcein.
Add the samples to the master mix.
Then, when the solution is put in a (dry lock) heat bath with a temperature of about 65 Â°C, the LAMP-reaction proceeds.
Due to the quenched calcein molecules which start to emit green fluorescence when the LAMP reaction proceeds, the presence of the target DNA can be detected. In most cases, the results can be judged by the naked eye. However, a hand-held ultraviolet lamp is more sensitive.
For a total overview of materials and procedures, see appendix.
The total length of the test (in minutes):
Collecting sample 1-2 minutes
DNA extraction 20-25 minutes
Amplification 40-60 minutes
Detection 1-2 minutes +
It seems a long time, though many DNA samples can pass the LAMP reaction at the same time. Also, when people work together, have proper tools and get more skilled in isolating DNA, the total length of the test will drastically decrease. Under normal conditions, a single person can test and give results to nearly 25 patients per day. However, when a team of three works together, this can run up to 80-90 patients per day.
Costs per test
Cotton swabs â‚¬0,05
Closable sputum cups â‚¬0,45
Costs per test Off costs
DNA extraction kit â‚¬0,10 â‚¬260,00 *
Costs per test Off costs
Buffer 2,5Î¼l â‚¬0,05
Primer mix 4,0Î¼l ** â‚¬45,00
BST 2.0 polymerase 1,0Î¼l â‚¬0,03
dNTP's 2,0Î¼l â‚¬0,28
MilliQ water 6,5Î¼l â‚¬0,01
1,5 ml EPJES â‚¬0,04
Heat bath â‚¬300,00
Costs per test Off costs
Staining mix 2,0Î¼l â‚¬0,04
Handheld UV lamp â‚¬48,00
The costs per test will vary between â‚¬1,00 and â‚¬1,20. Considering the off costs, it's better to look at the average costs of a large number of tests.
E.g. the price of one test out of thousand: â‚¬1,10 + â‚¬0,65 = â‚¬1,75
* A fairly expensive DNA extraction kit is faster, more reliable and more effective. The costs per test include different kinds of surfactants, detergents and alcohols (all in very small concentrations).
** Primer design is a difficult and expensive process. However, the incorporation of the enzyme reverse transcriptase can be combined with traditional PCR to allow for the amplification of the primer. This means that the primers would have to be purchased once. Only the dNTP's must be repeatedly acquired. The costs per test for the primer mix depend on the number of tests executed, because primers can be amplified to large numbers with relatively low costs.