Dr. Dan Cojoc, Optical Manipulation Lab @ IOM-CNR, Trieste, Italy
Research performed within the international collaboration with the groups of:
Prof. Zeev Zalevsky, Bar-Ilan University, Israel
Dr. Vicente Mico, University of Valencia, Spain
Malaria is an old disease but still claims almost a million deaths a year. This disease is caused by a parasite called Plasmodium, which is transmitted via the bites of infected mosquitoes.
In the human body, the parasites multiply in the liver, and then infect red blood cells (RBC). The life cycle of the parasite within the RBC is 48 hours. Therefore the diagnosis of malaria must be rapid, accurate, simple to use, portable and low cost, as suggested by the World Health Organization (WHO). Despite recent efforts, the gold standard remains the light microscopy of a stained blood film (Giemsa). This method can detect low parasitemia and identify different species of Plasmodium. However, it is time consuming, requires well trained personnel and good instrumentation that cannot be easily transported and installed everywhere. Therefore the costs become considerable and fast and accurate diagnosis impractical in remote areas as those of the African continent.
The aim of our research is to overcome these shortcomings analysing the thermal vibration of the cells by an optical technique. When the parasite enters a RBC, the mechanical properties of the cell e.g. cell stiffness and deformability change too.
These changes make an infected cell vibrate in a different way then a healthy cell. These are thermal vibrations with the bandwidth in the acoustic range, which means the cell emits a sound like signal. The core of our work is how to listen to this sound and interpret it to discriminate between infected and healthy cells.
We developed an optical method based on the temporal variation of the speckle formed by a RBC illuminated with a tilted laser beam to sense the dynamics of cell vibration or in other words, to see “the sound of Malaria”. Once the sound is recorded, proper data processing allows to separate the bad sound from the good sound and thus automatically separate the infected RBCs.
Preliminary results from our study allow to estimate that diagnosis of Malaria can be performed in half an hour, using just a drop of blood with a mini-microscope that can be transported and easily operated everywhere.
Malaria is an infectious disease caused by the bite of a female anopheles mosquito infected with Plasmodium. Every year, 243 millions of new cases are reported by the World Health Organization (WHO) with almost 1 million deaths, mostly African children.
In other words this means that every minute two children dye by Malaria in the world. Due to the nonspecific symptoms (fever) and the absence of a rapid diagnostic tool, presumptive anti-malarial treatment is often preferred. This increases the risk of mortality due to inappropriate therapy, and favors the emergence of drug resistance. Thus, new tools for a prompt and accurate malaria diagnosis are urgently needed.
The ideal diagnostic tool for malaria in endemic countries must be rapid, accurate, simple to use, low cost and easly interpretable .
The gold standard in malaria diagnosis is the microscopy of a smear blood film (Giemsa-stain), introduced by the German microbiologist Gustav Giemsa almost a century ago. Giemsa stain attaches specifically to regions of DNA of the parasite.
As DNA can be present only in nucleus and RBCs do not have a nucleus, the presence of Giemsa stain indicates the presence of the Malaria parasite inside the RBC. In spite of different efforts to find new techniques, Giemsa-stain still remains the only method allowing detection with high sensitivity and specificity. The life cycle of the Malaria parasite inside the RBC is about 48 hours and is summarized in the figure below by images of different stages of the infected RBC stained with Giemsa. The part in violet-indigo colour correspond to the parasite which evolves after cell invasion from ring to throphozoite and then schizont. After other few ours the cells explodes and many replica of the parasite are diffused into the blood vessel being ready for another, amplified, invasion.
Fig. 1 Life cycle of the parasite inside a RBC
Low parasitemia (5 to 10 parasites/µl of blood, i.e. about 0.0001% parasitemia) detection is possible by Giemsa stain. It allows also to identify the parasite species (P. falciparum, P. vivax, P. malariae, P. ovale) . Note that even if 1 µl (1 mm3) seems a small volume, the number of RBCs contained in this volume is large (about 5 millions cells / µl for normal hematocrit). Let us now consider just a small spherical drop of blood with a diameter of 4 mm. Its volume is a bit more than 30 µl and contains about 150 millions of RBCs to be analyzed. For a very low parasitemia, which however indicates the presence of the Malaria parasite in the patient’s blood, one has to identify about 150 infected cells over a total of 150 millions cells to have a good statistics. This requires a qualified microscopist and takes time because the technique cannot be automatized. Misinterpretation (artifacts mistaken for malaria parasite as fungi, bacteria or cell debris) can commonly occur in poor setting laboratories with low quality microscopes where only the experience of a well trained microscopist can reduce the errors. The time to diagnosis is about 8-10 h in African medical centers. The cost of equipment and training is considerable, even if the apparent cost for an individual sample examination is relatively low. Moreover, the equipment cannot be easily transported and installed. An improvement of diagnostic tools is in this context highly demanded. New innovative technologies could be used to enhance the accuracy while reducing time, complexity and cost of actual diagnosis.
Parasite development continuously remodels the membrane and cytoskeleton of the host RBC. Modification of more than 100 proteins of the host RBC proteome that could have an important impact on the morphology and rheological properties of the infected RBC (iRBC) and on malaria pathogenesis have been recently reported . Most of them are exported to the surface making the iRBC more adhesive and promoting thereby cytoadherence. Progressive stiffness of the iRBC membrane is also reported . Stiffness influences deformability, which is mildly reduced in rings (increased sphericity) and markedly altered in schizonts. Molecular biology and proteomics are largely used to investigate the membrane structures in both normal/healthy and parasitized RBC . However, these techniques cannot fully explain the biomechanical modifications (such as elasticity, deformability and stiffness) occurring during malaria infection. Therefore, a complete separation of iRBC from normal or uRBC by biochemical and biomolecular technique may have some limits.
The techniques presently available to measure biomechanical changes of the cells can be classified in: microfluidics and optical techniques.
Microfluidics is based on recent progresses in micro and nanofabrication techniques and use of new materials, which make possible to create microchannels and flows with properties similar to that of the blood vessels. The margination effect, based on the deformability of RBCs, has recently been used for separation of malaria iRBCs . This technique employs a simple microfludic channel through which RBCs are flowed. Stiffer cells tend to go toward the lateral walls of the channel and thus can be collected in proper reservoirs at the end of the channel. Interestingly, the separation effect is maximum in 40 % normal hematocrit, allowing to process a large number of cells / unit of time. However, this technique is limited to 90 % recovery for late stage (thropozoite) and 75 % recovery for early stage (ring) iRBCs. The performance, in terms of accuracy, is still far from Giemsa stain but can be used as an iRBCs pre-concentration approach.
Alternatively, optical techniques are mainly based on novel diffraction microscopy schemes, made possible by the development of new laser sources and fast and sensitive detectors. Diffraction phase microscopy has been proposed for an accurate characterization of the dynamics of the cell membrane, and hence its stiffness, and the composition of the cell . Measuring the stiffness from the thermal fluctuations of the membrane, Park et al present the comparative results for uRBC and iRBC at different erythrocytic stages . The stiffness varies from by a factor of ten from healthy to infected RBC (6 μN/m for uRBC, 14 μN/m for ring and 72 μN/m for schizont). While adequate for single cell analysis this method is too slow for diagnosis and its implementation requires an expensive instrumentation which cannot be easily transported. Therefore we were trying to apply a simpler and faster optical technique, called secondary speckle sensing microscopy, to detect RBC infected by P.f. malaria parasite.