Team:JMU Wuerzburg/Applied Design
Background – an introduction to Malaria
Malaria is an infectious disease spread worldwide that causes an estimated
number of 400.000 deaths per year according to the World Malaria Report 2017.
Mostly young children in Sub-Saharan Africa are affected that severely.
It is caused by certain protozoan parasites of the genus Plasmodium whose
primary hosts are Anopheles mosquitos. Through bites of these mosquitos,
Malaria is transmitted to humans and other animals. 200 million new cases of
malaria are reported every year by the WHO. The number of incidences is declining,
but still despite the enormous expenses, a global agenda to control and eradicate
malaria remains unfinished. 1
In humans, malaria is caused by five parasite species which all belong to the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. The most common are Plasmodium vivax and Plasmodium falciparum, which are responsible for the most severe cases of malaria.
There are several problems that complicate the mission to control Malaria. The ability of the pathogen to develop resistance to medical treatment is just one example. Due to these resistances the need of new drugs and clinical approaches is as urgent as never. 2
The most common method in malaria diagnostics is based on the microscopic examination of blood smears. The result is largely dependent on the skill of the person examining the blood and on the present levels of parasite. Although antigen- and PCR-based tests exist, they are often too expensive or not manageable due to the lack of appropriate equipment. Also, in respect of the rising resistances, a highly reliable, cheap and easily usable diagnostic method is needed to prescribe the right drug and to avoid incorrect, since delivering the correct diagnosis as soon as possible after infection is essential for a successful trial. 3
In humans, malaria is caused by five parasite species which all belong to the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. The most common are Plasmodium vivax and Plasmodium falciparum, which are responsible for the most severe cases of malaria.
There are several problems that complicate the mission to control Malaria. The ability of the pathogen to develop resistance to medical treatment is just one example. Due to these resistances the need of new drugs and clinical approaches is as urgent as never. 2
The most common method in malaria diagnostics is based on the microscopic examination of blood smears. The result is largely dependent on the skill of the person examining the blood and on the present levels of parasite. Although antigen- and PCR-based tests exist, they are often too expensive or not manageable due to the lack of appropriate equipment. Also, in respect of the rising resistances, a highly reliable, cheap and easily usable diagnostic method is needed to prescribe the right drug and to avoid incorrect, since delivering the correct diagnosis as soon as possible after infection is essential for a successful trial. 3
Motivation – improving diagnosis
We want to contribute to the improvement of Malaria diagnosis.
Rapid and correct diagnosis would save valuable time for an appropriate
medical treatment. A new diagnostic tool that enables correct, rapid and specific
detection of plasmodium in the human blood could compensate for deficits of
the current detection methods. In doing so we address the most urgent problems
that tend to prevent a large-scale application of diagnostic test systems
in countries with a poorly developed health care system. In fact,
P. falciparum is responsible for most of the severe cases of Malaria
with the highest mortality.4 A test system to distinguish between P.
falciparum and other species could quickly provide useful information to
select the correct drug for a more targeted therapy. We hope that our effort
helps to progress towards a world with less people suffering from Malaria.
Design
Benefits
We propose a design of a detection system that is easy
to use without any medical training and does not require
expensive technical equipment. It is not dependent on a reliable
power supply. Consequently, the application of the test system is
not restricted to medical facilities. It enables diagnostic testing
directly at the bedside of the patient with suspected Malaria.
The detection system indicates if a patient is suffering from Malaria.
Since Plasmodium falciparum is responsible for the most severe cases
of malaria, but not the only Plasmodium species causing human malaria,
our diagnostic tool specifically detects the DNA of these species.
Testing procedure and Hardware
Our test system relies on the detection of DNA of the parasite Plasmodium.
Many of the erythrocytes of an infected patient are filled with thousands
of copies of the parasite’s genome. At first, a blood sample
from a patient with suspected malaria is taken.
The next steps are performed in a simple test tube with separated chambers to isolate different reactions. Through lysis of the blood cells the DNA is extracted. Afterwards the parasites DNA is purified and prepared for amplification. The isolated DNA is then amplified and detected by multiplex qPCR. For the amplification of characteristic parts of the Plasmodium genome we developed two pairs of primers. One of these indicates if a patient is affected by Plasmodium in general. The other one specifically detects the species Plasmodium falciparum. We use our BioBrick which contains a part of synthetic template for a conserved sequence in the genome of all human-pathogenic plasmodium species as a positive control to establish our qPCR.
The next steps are performed in a simple test tube with separated chambers to isolate different reactions. Through lysis of the blood cells the DNA is extracted. Afterwards the parasites DNA is purified and prepared for amplification. The isolated DNA is then amplified and detected by multiplex qPCR. For the amplification of characteristic parts of the Plasmodium genome we developed two pairs of primers. One of these indicates if a patient is affected by Plasmodium in general. The other one specifically detects the species Plasmodium falciparum. We use our BioBrick which contains a part of synthetic template for a conserved sequence in the genome of all human-pathogenic plasmodium species as a positive control to establish our qPCR.
Outlook
To avoid the need of expensive thermocyclers,
the amplification could be conducted isothermally by using RPA
(Recombinase Polymerase Amplification),
which is capable of amplifying very low initial concentrations of
DNA in around 20 minutes.5
The amplified DNA product could later be detected in a
lateral flow assay like is has been performed by Kersting et al. in 20146.
The presence of Plasmodium DNA and specifically Plasmodium falciparum DNA is indicated through a visible color signal.
Proof of concept
We tested the primers by means of synthetic DNA oligos,
genomic sequences in plasmids (our BioBrick) and genomic DNA
of Plasmodium falciparum in a qPCR setting. Multiplexing was also achieved.
List of References
1 http://www.who.int/malaria/media/world-malaria-report-2017/en2 http://www.who.int/malaria/areas/treatment/drug_efficacy/en/
3 http://www.who.int/malaria/areas/treatment/overview/en/
4 http://www.who.int/malaria/publications/atoz/9789241563697/en/
5 https://www.ncbi.nlm.nih.gov/pubmed/27160000
6 Kersting et al.: Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis (2014)