Wavelength Markers for Malaria ( Plasmodium Falciparum ) Infected and Uninfected Red Blood Cells for Ring and Trophozoite Stages

Malaria parasite, Plasmodium falciparum, uses haemoglobin in host red blood cells (RBCs) as a major source of nutrient in ring and trophozoite stages. This brings about changes in the morphology and functional characteristics of the RBCs. We investigate malaria infected RBCs and uninfected RBCs-ring and trophozoite stages using multispectral imaging technique. Four spectral bands were found to be markers for identifying infected and uninfected RBCs: 435 nm and 660 nm were common markers for the two stages whiles 590 nm and 625 nm were markers for the ring and the trophozoite stages respectively. These four spectral bands may offer potential diagnostic markers for identifying infected and uninfected RBCs, as well as distinguishing ring and trophozoite stages.


Introduction
Malaria parasites, Plasmodium falciparum (P.falciparum), found in RBCs, remain the major contributor to morbidity and mortality especially in poor countries (Sachs & Melaney, 2002;Hay, Guerra, Tatem, Noor, & Snow, 2004;Paton et al., 2011).According to the World Health Organization (WHO), malaria parasites cause more than 1 million deaths with many cases of infections reported annually (Makler, Palmer, & Ager, 1998;Ross, Pritchard, Rubin, & Duse, 2006;Tek, Dempster, & Kale, 2009;Savkare & Narote, 2011).Infants and children account for most of the mortality from malaria and the disease accounts for ninety percent of the deaths of children in Africa (Amexo, Tolhurst, Barnish & Bates, 2004).Mortality caused by malaria parasites in most rural areas in Africa remains high because there is inadequate access to treatment (Weatherall et al., 2002).
Generally, the P. falciparum has a complex life cycle that consists of an asexual phase in the host and a sexual phase in the mosquito.The erythrocytic asexual phase involves the merozoites entering into the RBCs to become the ring stage.At the ring stage the parasite can transform to trophozoite and subsequently schizont or gametocyte after several cycles (Arora, Park, Mauser, Chakrabarti, & Schulte, 2011).The determinant of this transformation is underpinned by the digestion of haemoglobin (Hb) by the parasite using specific enzymes.
Studies have suggested that the discriminating spectral bands for infected RBCs (iRBCs) and uninfected RBCs (uRBCs) are found in the region 590-700 nm (Omucheni, 2012).Combined micro-Raman and micro-absorption techniques also show a comparative analysis of the iRBCs and uRBCs in the UV-visible region (Arora et al., 2011).Furthermore, an explanation model based on core-shell Mie scattering calculations presented the analysis of the iRBCs and uRBCs in the UV-visible region (Yulia, Patel, & Garcia-Rubio, 2010).These opinions therefore calls for further studies in order to identify the specific spectral band(s) that can be used as a marker(s) for iRBCs and uRBCs.Discriminating the stage of the malaria parasite will help in precise diagnosis and direct control of their development (Moore et al., 2006).

Samples
Samples used for this research work were cultured RBCs, with both infected and uninfected cells, stained with Giemsa dye on thin film slides, and grouped into ring and trophozoite stages.The cultured RBCs were obtained from ten (10) individuals, each contributing ten (10) slides each for a stage.These slides were obtained from the Department of Biomedical and Forensic Sciences, School of Biological Sciences, University of Cape Coast (UCC).

Multispectral Imaging
Multispectral images of the thin blood smear slides were acquired in transmission mode, using Multispectral Light Emitting Diodes Imaging Microscope system (MSLEDIM), which employed nine light emitting diodes (LEDs), as implemented in Brydegaard et al. (Brydegaard, Merdasa, Jayaweera, Ålebring, & Svanberg, 2011).The nine LEDs emit a total of thirteen spectral bands for the system.One of the LEDs is a triple-band (470 nm, 525 nm and 810 nm) and two others are dual-bands (375 nm, 400 nm and 625 nm, 850 nm) whiles the remaining six are of single-bands (435 nm, 590 nm, 660 nm, 700 nm, 750 nm, and 940 nm).A LabVIEW (National Instrument, NI) program is used to control the LEDs and the image acquisition.In acquiring images, the 590 nm spectral band is used to adjust the camera settings, (i.e. the gain and the exposure time).The images of the thin blood smear slides were acquired in grayscale and saved in tagged image file format (TIFF) for each of the thirteen spectral bands.The images were imported into a MATrix LABoratory (Matlab) (R2010a Matlab 7.10.0,Mathworks Inc.) platform and the pixel intensities representing iRBCs and uRBCs, were extracted.

Principal Component Analysis
Principal Component Analysis (PCA) was performed on the intensity data using Matlab algorithm.PCA is a non-parametric method of extracting relevant information from the multidimensional data set.The reduction of the complex multidimensional data set to a lower dimension reveals the principal hidden or latent factors.
Data set, X of m observations and n variables is centered X c such that the elements of the matrix of dimension m×n are around the sample mean of zero.The X c is then converted into covariant matrix S defined as where X c ' denotes the matrix transpose of X c .This square, symmetric, nonsingular covariant matrix S is further transformed into a diagonal matrix using the relation below, where the columns of β contain the eigenvectors of S and M is a diagonal matrix with the eigenvalues along the diagonal.β is used to obtain new variables called Principal Components (PCs).
Two sets of PCA analysis were done; one with all the 13 variables (spectral bands) and 252 observations (intensities) (252×13 data points), the second was performed with ten (10) variables (ten unique individuals).Scree plots were used to determine the number of principal components (PCs) appropriate for discrimination and classification.The PCA reduced the dimensionality of the observations, which are the intensity values at the various spectral bands.

Results and Discussions
Figure 1a shows the grayscale image of a thin blood smear slide sample with the 590 nm LED source.The image shows a bright background interspersed with spots representing iRBCs and uRBCs.This spectral band was used as a standard due to the camera's sensitivity to it.Figures 1b-d represent grayscale images of the same thin blood smear slide sample with 435 nm, 700 nm, and 940 nm LED sources respectively.The images have the same dimensions but different grayscales.The other images (not shown) have the same dimensions as in Figure 1 and also show different grayscales.The different grayscales are as a result of selective spectral band absorption of the LED sources.trophozoite stages of iRBCs and uRBCs.Whilst 590 nm and 625 nm spectral bands are distinct markers for ring and trophozoite stages of iRBCs and uRBCs respectively.This is an indication that the iRBCs more or less have their Hb either partially or completely digested together with the membrane proteins and membrane skeletal proteins by the parasite.The iRBCs therefore become less dense hence able to transmit more light (Goldberg, Slater, Cerami, & Henderson, 1990;Moore et al., 2006).The intensity differences may be due to the breakdown of Hb by the parasite, the protein chain fragments are transported away for further digestion.Also absorption curves of haemoglobin (Hb) have strong bands in the visible part of the electromagnetic spectrum.The molar extinction coefficient for Hb is very high from 405-450 nm (Soret band), and 660 nm.For various parasitic stages (e.g.Ring and Trophozoite ), the presence of hemozoin or the decrease of Hb would increase transmission.Therefore, incident radiation in this range will be very highly absorbed by cells with high concentration of Hb.Generally, transmitted light intensities of iRBCs were higher than that of uRBCs.The first two coefficients of PC1, which represents the greatest amount of the variation in the sample and overall average light intensities detected, are considerably larger in magnitude suggesting that PC1 was dominated by two spectral bands.In Figure 4, the PC1 scores are between -200 and -400 and since PC1 has the highest variability, this indicates the consistency of uRBCs characteristics.In Figures 5, 6, 7, and 8 we show the 3D scatter plots of the first three PCs which comprises of ten variables each (ten individuals) obtained from 435 nm 660 nm, 470 nm, and 700 nm spectral bands respectively for iRBCs and uRBCs at the two stages of the parasite.
In Figures 5 and 6, there is a discrete separation pattern between iRBCs and uRBCs for both ring stage and trophozoite stage.The 3 PCs (PC1, PC2, and PC3) of uRBCs have negative scores, while the iRBCs have its negative scores only in PC3.From Figure 7, the iRBCs at both ring and trophozoite stages cannot be discriminated, that is there is no discrete separation pattern between iRBCs and uRBCs at the 470 nm spectral band.A similar trend is seen in Figure 8 for 700 nm spectral band, there is no discrete separation pattern between iRBCs and uRBCs for both stages.

Conclusions
Using transmitted intensities extracted from multispectral images, four spectral bands: 435 nm, 590 nm, 625 nm and 660 nm have been identified to be markers for distinguishing infected red blood cells (iRBCs) and uninfected red blood cells (uRBCs) in ring and trophozoite stages of P. falciparum parasite.The 435 nm and 660 nm spectral bands were found to be common markers for the two stages whiles 590 nm and 625 nm spectral bands were markers for the ring and trophozoite stages respectively.With principal component analysis (PCA), which accounted for 98.9% of the total variation of the transmitted light intensities, 3 PCs confirmed these spectral bands as markers in the scatter plots.These markers offer a potential diagnostic tool for discriminating iRBCs and uRBCs, as well distinguishing ring and trophozoite stages.The analysis may be implemented in the field by any trained personnel.

Figure 1 .
Figure 1.Some of the grayscale images captured from the thin blood smear slides with ring stage of P. falciparum using the Multispectral Light Emitting Diodes Imaging Microscope system at (a) 590 nm (b) 435 nm, (c) 700 nm and (d) 940 nm spectral bands.The spots in the images represent the iRBCs and the uRBCs

Figure 2 .
Figure 2. The maximum grayscale pixel intensities extracted from the images of the slides with (a) ring stage and (b) trophozoite stage of P. falciparum infected RBCs (iRBCs) and uninfected (uRBCs).The common spectra bands have been indicated with a black ring and the independent spectra band with yellow ring (Ring stage) and green ring (trophozoite stage)

Figure 3
Figure3depicts the scree plot for the thirteen (13) variables and 252 observations, which was used to select the principal components (PCs) that best explain the transmitted intensities obtained from the images.Three PCs retain the maximum variance of 98.9% cumulative variability of the intensity data.Out of the 98.9%, PC1, PC2 and PC3 contributed 64.9%, 31.0%and 3.0% variability respectively.The PCs are proportional to the transmitted intensities by the iRBCs and uRBCs.The first PC (PC1) is the linear combination of the intensity data with maximum variance, PC2 is the linear combination of the next maximum variance orthogonal to PC1.PC3 is the linear combination of the third maximum variance orthogonal to both PC1 and PC2 in that order.

Figure 4 .
Figure 4. Scatter plot of the first three principal components from the scree plot (Figure 3) revealing iRBCs at ring stage( ), iRBCs at trophozoite stage( )and uRBCs( ) for all the thirteen (13) spectral bands

Figure 5 .
Figure 5. Scatter plot of the first three principal components of (a) ring stage and (b) trophozoite stage of P. falciparum infected RBCs (iRBCs) and uninfected RBCs (uRBCs) at 435 nm spectral band

Figure 8 .
Figure 8. Scatter plot of the first three principal components of (a) ring stage and (b) trophozoite stage of P. falciparum infected RBCs (iRBCs) and uninfected RBCs (uRBCs) at 700 nm spectral band