Scandinavian Journal of Infectious Diseases, 2014; 46: 368–375
ORIGINAL ARTICLE
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A morphology-based method for the diagnosis of red blood cells parasitized by Plasmodium malariae and Plasmodium ovale Alireza Karimi1,2, Mahdi Navidbakhsh1,2, Afsaneh Motevalli Haghi3 & Shahab Faghihi4 From the 1School of Mechanical Engineering, Iran University of Science and Technology, 2Tissue Engineering and Biological Systems Research Laboratory, School of Mechanical Engineering, Iran University of Science and Technology, 3Medical Parasitology and Mycology Department, Tehran University of Medical Sciences, 4Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Abstract Background: The morphology of red blood cells (RBCs) is altered significantly during the maturation stages of malaria parasites, which include ring, trophozoite, and schizont. There is dissimilarity in terms of the morphological characteristics of parasitized RBCs infected by the 4 species of Plasmodium, including falciparum, vivax, malariae, and ovale. This makes the process of diagnosis very difficult, which may lead to a wrong treatment method and substantial damage to the health of the patient. An innovative technique in introduced that accurately defines the shape of parasitized RBCs at each stage of infection as a potential method of diagnosis. Methods: Giemsa-stained thin blood films were prepared using blood samples collected from healthy donors as well as patients infected with P. malariae and P. ovale. The diameter and thickness of healthy and infected RBCs at each stage of infection were measured from their optical images using Olysia and Scanning Probe Image Processor (SPIP) software, respectively. A shape equation was fitted based on the morphological characteristics of RBCs, and their relative 2-dimensional shapes were plotted using Wolfram Mathematica. Results: At the ring stage, the thicknesses of RBCs parasitized by P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) increased by 42% and 51%, respectively. Both Pm-RBCs and Po-RBCs remained nearly biconcave throughout parasite development even though their volumes increased. Conclusions: It is proposed that the morphology-based characterization technique introduced here could be used to intensify the accuracy of the Giemsa staining diagnosis method for the detection of the Plasmodium genus and infection stage. Based on the significant morphological alterations induced by different Plasmodium species, the results may also find practical use for faster prediction and treatment of human malaria.
Keywords: Malaria, red blood cell, morphological characterization, shape equation, Plasmodium malariae, Plasmodium ovale
Introduction Malaria is the world’s most prevalent vector-borne disease and is caused by peripheral blood parasites of the genus Plasmodium. It remains among the most devastating diseases, responsible for half a billion infections globally leading to a mortality of 2–3 million every year [1–3]. Malaria is caused by 4 species of Plasmodium, with Plasmodium falciparum and Plasmodium vivax being the most widespread. P. falciparum causes the most severe form of human malaria and is responsible for almost all
mortalities, while P. vivax is the most omnipresent human malaria parasite and leads to significant morbidity and socioeconomic problems [4–6]. In addition, Plasmodium malariae and Plasmodium ovale are found at very low prevalence in East Asia, as well as in areas with intense malaria transmission. They are also widely distributed in most tropical regions of Africa, Asia, and South America [7,8]. As P. malariae and P. ovale are both considered to be uncommon, mild in clinical presentation, and
Correspondence: S. Faghihi, Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran. Tel: 98 21 44580461. Fax: 98 21 44580386. E-mail: [email protected], [email protected] (Received 7 October 2013; accepted 24 December 2013) ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2014.880186
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easily treatable with the conventional antimalarial drug chloroquine, there is shortage of knowledge about these less common human malaria parasites [7]. There are substantial similarities in the morphologies of P. ovale and P. vivax [7], as well as P. malariae and P. falciparum [9] when they are fixed and stained. This makes the process of identification very difficult and may result in the wrong treatment procedure. The common method of diagnosis for P. ovale and P. malariae is based on the examination of peripheral blood films stained with Giemsa dye. Giemsa staining is considered the gold standard method for the diagnosis of parasitized red blood cells (RBCs) [10,11]. However, it is not possible to identify the differences between the 4 species of malaria parasite, as well as their stages of infection, with Giemsa staining [12–14]. It is known that differentiation between the human malaria parasites, particularly P. vivax and P. falciparum, is difficult by Giemsa staining [15–17]. The wrong diagnosis could lead to a marked underestimation of the burden of illness, exacerbate the illness [9], and carry a high rate of mortality [18]. Thus, accurate diagnosis of the malaria parasite is essential for the appropriate treatment of malaria [15]. The RBC is central to the malaria lifecycle. The invasion and growth of the malaria parasite inside the healthy RBC (HRBC) cause irreversible changes in its morphology and biophysical characteristics [19,20]. A limited number of studies have been conducted on the changes that occur when erythrocytes are infected with P. ovale and P. malariae [21–23]. These changes appear to be similar to those seen with the other species of Plasmodium, particularly P. vivax and P. falciparum. A method that determines the morphological characteristics of healthy RBCs and those parasitized by P. falciparum and P. vivax has been proposed previously using an optical imaging technique [24]. The shape of parasitized RBCs is transformed from biconcave to near spherical during the maturation of human malaria parasites. It is possible to quantitatively characterize the morphological changes in parasitized RBCs, and this characterization may lead to a better understanding of the pathophysiology, parasite maturation, and interaction of parasites with host RBCs, and most importantly to the development of a new method of diagnosis for identifying and treating this fatal disease. In the present study, the precise shapes of RBCs parasitized with P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) were determined at each stage of parasite maturation and compared with P. falciparum (Pf-RBCs) and P. vivax (Pv-RBCs). Giemsa staining was used as the most common and known protocol in order to prepare test specimens
An innovative morphology-based diagnosis method 369 from blood samples collected from patients with P. malariae, P. ovale, P. vivax, and P. falciparum. Materials and methods Blood samples of African patients with P. malariae (n 8) and P. ovale (n 9) malaria were used in order to modulate a robust shape equation for infected RBCs (IRBCs) from the early ring stage to the late schizont stage. Test specimens were prepared by centrifuging the patient’s blood at 1000 g for 5 min. After removal of the plasma, the RBCs were washed in RPMI 1640 and thin blood films were prepared by Giemsa staining. Blood samples from healthy donors (n 10) were used as controls. Finally, the thin blood films of the Pm-RBCs and Po-RBCs were imaged using an optical microscope (Olympus BX41, with Olysia software; Olympus Corporation, Tokyo, Japan) with 100/1.40 oil objective. Structural measurements and modeling The morphological characteristics of healthy and parasitized RBCs were obtained using a previously described method [24]. The final shape equation is represented by equation 1.
(
(Y b )2 X 2
ae
) ae((Y c )
2
X2
) d
(1)
Mathematical modeling resulted in the above shape equation, which consists of 4 constant coefficients a, b, c, and d. These constant parameters are obtained based on fitting of the mathematical model to the morphological characteristics including diameter (D) and 2 thicknesses (t1 and t2) of healthy RBCs (HRBCs) and IRBCs. Finally, 2-dimensional (2D) shape equations of RBCs at each stage of infection were drawn using Wolfram Mathematica version 8 (Wolfram Research, Champaign, IL, USA).
Figure 1. Diagram of a red blood cell showing its morphological characteristics.
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370 A. Karimi et al.
Figure 2. Optical micrographs of Giemsa-stained blood films as well as SPIP images of red blood cells infected by Plasmodium malariae, presenting the 3 stages of infection: ring (a, a1), trophozoite (b, b1), and schizont (c, c1).
Figure 3. Optical micrographs of Giemsa-stained blood films as well as SPIP images of red blood cells infected by Plasmodium ovale, presenting the 3 stages of infection: ring (a, a1), trophozoite (b, b1), and schizont (c, c1).
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Results and discussion The morphological characteristics measured were the diameter (D), thickness at the thickest part of the RBC (t1), and central thickness (t2), as shown in Figure 1. Optical micrographs of Giemsa-stained blood films as well as Scanning Probe Image Metrology A/S, Hørsholm, Denmark Processor (SPIP;) images of RBCs infected by P. malariae are shown in Figure 2. The figure shows the RBCs parasitized by P. malariae at the 3 stages of infection, including ring, trophozoite, and schizont. Figure 3 shows the same images for RBCs parasitized by P. ovale. The SPIP images representing the Z-range for both P. malariae and P. ovale infected RBCs obtained from thin blood smear are shown in Figure 4. The results showed that the Z-range of parasitized RBCs at the schizont stage increased 2.40 times for Pm-RBCs and 2.69 times for Po-RBCs compared to the HRBCs. Interestingly,
Figure 4. SPIP images plus Z-range of red blood cells infected by Plasmodium malariae (a) ring, (b) trophozoite, and (c) schizont, as well as Plasmodium ovale (d) ring, (e) trophozoite, and (f) schizont.
An innovative morphology-based diagnosis method 371 the diameter (D) of the Po-RBCs increased more significantly during parasite maturation (ring to schizont) compared to the Pm-RBCs (Figure 5), whereas the thicknesses (t1 and t2) increased similarly for both Pm-RBCs and Po-RBCs as the intraerythrocytic parasite grew. Table I shows the diameter (D), thicknesses (t1 and t2), and Z-range pixels of HRBCs, Pm-RBCs, and Po-RBCs. Using the parameters D, t1, and t2, a general 2D shape equation is fitted (equation 2). (
(Y b )2 X 2
ae
) ae((Y c )
2
) d ,(x range, y range)
X2 )
(2)
In order to obtain the constant coefficients a, b, c, and d for Pm-RBCs and Po-RBCs, the shape equations at each stage of infection were fitted to the parameters D, t1, and t2. Table II shows the constant parameters along with the related shape equations. Based on the shape equations it was possible to plot the shape of infected RBCs at each stage of infection. The results indicated that Pm-RBCs at the ring, trophozoite, and schizont stages had diameters of 7.89, 8.23, and 8.39 mm, respectively. Our previous study indicated that Pf-RBCs had diameters of 7.92, 8.28, and 8.47 mm at the ring, trophozoite, and schizont stages, respectively. The Po-RBCs showed diameters of 8.86, 9.86, and 10.96 mm, which are similar to those of Pv-RBCs at 8.90, 9.52, and 10.86 mm respectively, for ring, trophozoite, and schizont stages [24]. This is in agreement with previous studies, which have reported almost equal diameters for Pm-RBCs and Pf-RBCs, as well as Po-RBCs and Pv-RBCs. This could make the process of malaria diagnosis very difficult, which may lead to the wrong treatment procedure. From the ring to subsequent schizont stage, Pm-RBCs and Po-RBCs retained their biconcave shape (69% and 77% increase in t2 for Pm-RBCs and Po-RBCs, respectively, compared to the ring stage) (Table I, Figure 5). It is known that at the ring stage, a Pv-RBC retains its biconcave shape while a Pf-RBC almost loses its biconcave shape (50% and 93.40% increase in t2 for Pf-RBCs and Pv-RBCs, respectively, compared to ring stage) [24]. This is the reason why Pv-RBCs at the ring stage can still circulate freely in the peripheral blood [25]. Figure 6 presents the 2D shapes of Pf-RBCs, Pv-RBCs, Pm-RBCs, and Po-RBCs at the trophozoite stage, measured and plotted using the shape equations, in order to facilitate the morphological comparison between RBCs infected with the 4 different species of Plasmodium. The results of the present study showed that as the parasite enlarges, the Pm-RBC retains its biconcave shape, whilst the Pf-RBC almost entirely loses its biconcave shape and becomes nearly spherical at the
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372 A. Karimi et al.
Figure 5. 2D shapes of red blood cells parasitized by Plasmodium malariae (a) ring, (b) trophozoite, and (c) schizont, as well as Plasmodium ovale (d) ring, (e) trophozoite, and (f) schizont.
An innovative morphology-based diagnosis method 373
Table I. Diameter, thickness, and Z-depth of healthy and parasitized red blood cells measured by Olysia and SPIP software.
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Stage Plasmodium malariae Z-depth (pixel)a (number of cells)b D (mm)c t1 (mm)c t2 (mm)c Plasmodium ovale Z-depth (pixel)a (number of cells)b D (mm)c t1 (mm)c t2 (mm)c
HRBC
Ring
Trophozoite
Schizont
45.00 5.13 (40) 7.38 0.36 3.35 0.16 2.92 0.14
64.12 3.08 (37) 7.89 0.17 4.77 0.28 4.14 0.21
78.98 6.12 (34) 8.23 0.31 5.87 0.39 5.11 0.35
108.25 2.43 (32) 8.39 0.29 8.04 0.38 7.02 0.34
45.00 5.13 (40) 7.38 0.36 3.35 0.16 2.92 0.14
68.33 4.13 (35) 8.86 0.52 5.08 0.42 4.43 0.36
96.25 3.58 (33) 9.86 0.36 7.13 0.52 6.21 0.32
121.12 5.61 (30) 10.96 0.45 9.01 0.43 7.85 0.46
SPIP, Scanning Probe Image Processor; HRBC, healthy red blood cell. shows the depth of each red blood cell measured using SPIP software. bNumber of cells tested at each stage of parasite maturation inside the red blood cells. cData are reported at a significance level of p 0.05. aZ-depth
trophozoite and subsequent schizont stage (8.47, 9.19, and 8.01 mm for D, t1, and t2, respectively) [24]. The Pv-RBCs and Po-RBCs, furthermore, retain their biconcave shape throughout parasite development even though their volumes increase significantly (8.14 and 7.33 mm for Pv-RBCs and 9.01 and 7.85 mm for Po-RBCs for t1 and t2 at the schizont stage, respectively) (Figure 5). It has also been reported that erythrocytes infected with P. vivax are elliptical; this is similar to the results found in the present study for P. ovale, in which RBCs were found to have an elliptical and elongated shape [24,26]. The morphological characteristics of the IRBCs presented here could explain the significant increase in viscosity of the blood flow in the microcircular channel at the trophozoite stage, while at the ring stage, a similar viscosity to the healthy state model is
reported [27]. There have been substantial efforts to simulate the rheological behavior of Pf-RBCs in order to fundamentally understand the pathology of the disease and to predict the increase in flow resistance at each parasitemia level. However, in these studies, HRBCs and IRBCs were considered biconcave and spherical, respectively [19,27,28]. Cell geometry plays the main role in modeling and hemodynamic studies, and the shape of the RBC needs to be as accurate as possible. Therefore, it is believed that the shape equations introduced here could be used in rheological modeling studies to explain the rheological alterations caused by morphological changes in IRBCs. Since there has been no study on the rheological changes induced by Pm-RBCs or Po-RBCs, the shape information will be of benefit for such investigations. Most importantly we believe that the SPIP imaging method used
Table II. Two-dimensional shape equations for the healthy and infected red blood cells. Plasmodium malariae
(
) 30e((Y 2.35)
2
χ2
) 5101 , (6,6), (4.38, 3) ) (
(
) 30e((Y 2.10 )
2
χ2
) 6102 , (6,6), (4.60, 3.60) ) (
(Y 0.97 )2 χ2
HRBC
30e
Ring
30e
Trophozoite
50e
Schizont
80e
(Y 1.05 )2 χ2
(
) 50e((Y 1.90 )
χ2
) 15103 , (6,6), (4.80, 4) ) (
(
) 80e((Y 1.70 )
χ2
) 4105 , (6,6), (5.60, 5) ) (
(Y 1.1)2 χ2
(Y 1.1)2 χ2
2
2
Plasmodium ovale
(
(Y 0.97 )2 χ2
HRBC
30e
Ring
75e
Trophozoite
84e
Schizont
105e
HRBC, healthy red blood cell.
(
(Y 1)2 χ2
2
) 75e((Y 2.50 )
(
2
(Y 0.95 )2 χ2
(
) 30e((Y 2.35)
(Y 1)2 χ2
χ2
) 84e((Y 2.20 )
2
) 105e((Y 2 )
2
χ2
) 5101 , (6,6), (4.38, 3) ) (
) 8102 , (6,6), (5.20,3.70) ) (
χ2
χ2
) 6104 , (6,6), (5.70, 4.40) ) (
) 1105 , (6,6), (6.10,5.10) ) (
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374 A. Karimi et al.
Figure 6. 2D shapes of red blood cells parasitized by Plasmodium falciparum (a), Plasmodium vivax (b), Plasmodium malariae (c), and Plasmodium ovale (d), at the trophozoite stage.
in this study should be considered a new, easy, and additional method for the imaging of malaria parasites in order to quickly diagnose the type and stage of infection. In conclusion, the intention of this work was to introduce a new and simple method that has potential clinical use in the prediction, diagnosis, and treatment of human malaria for the clinicians, researchers, engineers, and technicians working on this disease. We believe that this method could be used to improve the accuracy of the Giemsa staining diagnosis method. We also hope that the results of this study will lead to a better understanding of the effect of morphological alterations on blood velocity and rheological changes induced by parasitized RBCs at each stage of infection. Acknowledgements This work was supported by the Iran University of Science and Technology and National Institute of
Genetic Engineering and Biotechnology. We gratefully acknowledge the participants for their cooperation and blood sample donation. We also thank the staff of the Mycology Department and Parasitology Laboratory of Tehran University of Medical Sciences for their collaboration. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Cisse B, Sokhna C, Boulanger D, Milet J, Bael H, Richardson K, et al. Seasonal intermittent preventive treatment with artesunate and sulfadoxine–pyrimethamine for prevention of malaria in Senegalese children: a randomised, placebo-controlled, double-blind trial. Lancet 2006;367: 659–67. [2] Williams TN. Red blood cell defects and malaria. Mol Biochem Parasitol 2006;149:121–7.
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[3] Chiyaka C, Mukandavire Z, Das P, Nyabadza F, Hove-Musekwa SD, Mwambi H. Theoretical analysis of mixed Plasmodium malariae and Plasmodium falciparum infections with partial cross-immunity. J Theor Biol 2010; 263:169–78. [4] Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria: neglected and not benign. Am J Trop Med Hyg 2007;77:79–87. [5] Zakeri S, Raeisi A, Afsharpad M, Kakar Q, Ghasemi F, Atta H, et al. Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3alpha and pvcsp genes as molecular markers. Parasitol Int 2009;59:15–21. [6] Patarroyo MA, Perez-Leal O, Lopez Y, Cortes J, Rojas-Caraballo J, Gomez A, et al. Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2. Biochem Biophys Res Commun 2005; 337:853–9. [7] Fuehrer HP, Habler VE, Fally MA, Harl J, Starzengruber P, Swoboda P, et al. Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in southern Asia. Int J Parasitol 2012; 42:693–9. [8] Guimarães LO, Bajay MM, Wunderlich G, Bueno MG, Röhe F, Catão-Dias JL, et al. The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil. Acta Trop 2012;124:27–32. [9] Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale—the ‘bashful’ malaria parasites. Trends Parasitol 2007;23:278–83. [10] Craig MH, Bredenkamp BL, Williams CH, Rossouw EJ, Kelly VJ, Kleinschmidt I, et al. Field and laboratory comparative evaluation of ten rapid malaria diagnostic tests. Trans R Soc Trop Med Hyg 2002;96: 258–65. [11] Ochola LB, Vounatsou P, Smith T, Mabaso ML, Newton C. The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis 2006;6:582–8. [12] Adeoye GO, Nga IC. Comparison of quantitative buffy coat technique (QBC) with Giemsa-stained thick film (GTF) for diagnosis of malaria. Parasitol Int 2007;56: 308–12. [13] Afzan MY, Sivanandam S, Kumar GS. Modified Field’s staining—a rapid stain for Trichomonas vaginalis. Diagn Microbiol Infect Dis 2010;68:159–62. [14] Clendennen TE III, Long GW, Baird JK. QBC® and Giemsastained thick blood films: diagnostic performance of laboratory technologists. Trans R Soc Trop Med Hyg 1995; 89:183–4.
An innovative morphology-based diagnosis method 375 [15] Kawamoto F, Liu Q, Ferreira MU,Tantular IS. How prevalent are Plasmodium ovale and P. malariae in East Asia? Parasitol Today 1999;15:422–6. [16] Kasehagen LJ, Mueller I, McNamara DT, Bockarie MJ, Kiniboro B, Rare L, et al. Changing patterns of Plasmodium blood-stage infections in the Wosera region of Papua New Guinea monitored by light microscopy and high throughput PCR diagnosis. Am J Trop Med Hyg 2006;75:588–96. [17] Zhou M, Liu Q, Wongsrichanalai C, Suwonkerd W, Panart K, Prajakwong S, et al. High prevalence of Plasmodium malariae and Plasmodium ovale in malaria patients along the Thai–Myanmar border, as revealed by acridine orange staining and PCR-based diagnoses. Trop Med Int Health 1998;3:304–12. [18] Eiam-Ong S. Malarial nephropathy. Semin Nephrol 2003;23:21–33. [19] Shelby JP, White J, Ganesan K, Rathod PK, Chiu DT. A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes. Proc Natl Acad Sci U S A 2003;100:14618–22. [20] Cranston HA, Boylan CW, Carroll GL, Sutera SP, Williamson JR, Gluzman IY, et al. Plasmodium falciparum maturation abolishes physiologic red cell deformability. Science 1984;223:400–3. [21] Aikawa M, Hsieh CL, Miller LH. Ultrastructural changes of the erythrocyte membrane in ovale-type malarial parasites. J Parasitol 1977;63:152–4. [22] Matsumoto Y, Matsuda S, Yoshida Y. Ultrastructure of erythrocytic stages of Plasmodium ovale in humans. Am J Trop Med Hyg 1986;35:689–96. [23] Matsumoto Y, Matsuda S, Yoshida Y. Ultrastructure of human erythrocytes infected with Plasmodium ovale. Am J Trop Med Hyg 1986;35:697–703. [24] Karimi A, Navidbakhsh M, Motevalli Haghi A, Faghihi S. An innovative shape equation to quantify the morphological characteristics of parasitized red blood cells by Plasmodium falciparum and Plasmodium vivax. Proc Inst Mech Eng H 2013;227:428–37. [25] Combes V, El-Assaad F, Faille D, Jambou R, Hunt NH, Grau GE. Microvesiculation and cell interactions at the brain–endothelial interface in cerebral malaria pathogenesis. Prog Neurobiol 2010;91:140–51. [26] Collins WE, Jeffery GM. Plasmodium ovale: parasite and disease. Clin Microbiol Rev 2005;18:570–81. [27] Serebrennikova YM, Patel J, Milhous WK, García-Rubio LH. Quantitative analysis of morphological alterations in Plasmodium falciparum infected red blood cells through theoretical interpretation of spectral measurements. J Theor Biol 2010;265:493–500. [28] Secomb TW. Mechanics and computational simulation of blood flow in microvessels. Med Eng Phys 2010;33: 800–4.
ORIGINAL ARTICLE
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A morphology-based method for the diagnosis of red blood cells parasitized by Plasmodium malariae and Plasmodium ovale Alireza Karimi1,2, Mahdi Navidbakhsh1,2, Afsaneh Motevalli Haghi3 & Shahab Faghihi4 From the 1School of Mechanical Engineering, Iran University of Science and Technology, 2Tissue Engineering and Biological Systems Research Laboratory, School of Mechanical Engineering, Iran University of Science and Technology, 3Medical Parasitology and Mycology Department, Tehran University of Medical Sciences, 4Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Abstract Background: The morphology of red blood cells (RBCs) is altered significantly during the maturation stages of malaria parasites, which include ring, trophozoite, and schizont. There is dissimilarity in terms of the morphological characteristics of parasitized RBCs infected by the 4 species of Plasmodium, including falciparum, vivax, malariae, and ovale. This makes the process of diagnosis very difficult, which may lead to a wrong treatment method and substantial damage to the health of the patient. An innovative technique in introduced that accurately defines the shape of parasitized RBCs at each stage of infection as a potential method of diagnosis. Methods: Giemsa-stained thin blood films were prepared using blood samples collected from healthy donors as well as patients infected with P. malariae and P. ovale. The diameter and thickness of healthy and infected RBCs at each stage of infection were measured from their optical images using Olysia and Scanning Probe Image Processor (SPIP) software, respectively. A shape equation was fitted based on the morphological characteristics of RBCs, and their relative 2-dimensional shapes were plotted using Wolfram Mathematica. Results: At the ring stage, the thicknesses of RBCs parasitized by P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) increased by 42% and 51%, respectively. Both Pm-RBCs and Po-RBCs remained nearly biconcave throughout parasite development even though their volumes increased. Conclusions: It is proposed that the morphology-based characterization technique introduced here could be used to intensify the accuracy of the Giemsa staining diagnosis method for the detection of the Plasmodium genus and infection stage. Based on the significant morphological alterations induced by different Plasmodium species, the results may also find practical use for faster prediction and treatment of human malaria.
Keywords: Malaria, red blood cell, morphological characterization, shape equation, Plasmodium malariae, Plasmodium ovale
Introduction Malaria is the world’s most prevalent vector-borne disease and is caused by peripheral blood parasites of the genus Plasmodium. It remains among the most devastating diseases, responsible for half a billion infections globally leading to a mortality of 2–3 million every year [1–3]. Malaria is caused by 4 species of Plasmodium, with Plasmodium falciparum and Plasmodium vivax being the most widespread. P. falciparum causes the most severe form of human malaria and is responsible for almost all
mortalities, while P. vivax is the most omnipresent human malaria parasite and leads to significant morbidity and socioeconomic problems [4–6]. In addition, Plasmodium malariae and Plasmodium ovale are found at very low prevalence in East Asia, as well as in areas with intense malaria transmission. They are also widely distributed in most tropical regions of Africa, Asia, and South America [7,8]. As P. malariae and P. ovale are both considered to be uncommon, mild in clinical presentation, and
Correspondence: S. Faghihi, Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran. Tel: 98 21 44580461. Fax: 98 21 44580386. E-mail: [email protected], [email protected] (Received 7 October 2013; accepted 24 December 2013) ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2014.880186
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easily treatable with the conventional antimalarial drug chloroquine, there is shortage of knowledge about these less common human malaria parasites [7]. There are substantial similarities in the morphologies of P. ovale and P. vivax [7], as well as P. malariae and P. falciparum [9] when they are fixed and stained. This makes the process of identification very difficult and may result in the wrong treatment procedure. The common method of diagnosis for P. ovale and P. malariae is based on the examination of peripheral blood films stained with Giemsa dye. Giemsa staining is considered the gold standard method for the diagnosis of parasitized red blood cells (RBCs) [10,11]. However, it is not possible to identify the differences between the 4 species of malaria parasite, as well as their stages of infection, with Giemsa staining [12–14]. It is known that differentiation between the human malaria parasites, particularly P. vivax and P. falciparum, is difficult by Giemsa staining [15–17]. The wrong diagnosis could lead to a marked underestimation of the burden of illness, exacerbate the illness [9], and carry a high rate of mortality [18]. Thus, accurate diagnosis of the malaria parasite is essential for the appropriate treatment of malaria [15]. The RBC is central to the malaria lifecycle. The invasion and growth of the malaria parasite inside the healthy RBC (HRBC) cause irreversible changes in its morphology and biophysical characteristics [19,20]. A limited number of studies have been conducted on the changes that occur when erythrocytes are infected with P. ovale and P. malariae [21–23]. These changes appear to be similar to those seen with the other species of Plasmodium, particularly P. vivax and P. falciparum. A method that determines the morphological characteristics of healthy RBCs and those parasitized by P. falciparum and P. vivax has been proposed previously using an optical imaging technique [24]. The shape of parasitized RBCs is transformed from biconcave to near spherical during the maturation of human malaria parasites. It is possible to quantitatively characterize the morphological changes in parasitized RBCs, and this characterization may lead to a better understanding of the pathophysiology, parasite maturation, and interaction of parasites with host RBCs, and most importantly to the development of a new method of diagnosis for identifying and treating this fatal disease. In the present study, the precise shapes of RBCs parasitized with P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) were determined at each stage of parasite maturation and compared with P. falciparum (Pf-RBCs) and P. vivax (Pv-RBCs). Giemsa staining was used as the most common and known protocol in order to prepare test specimens
An innovative morphology-based diagnosis method 369 from blood samples collected from patients with P. malariae, P. ovale, P. vivax, and P. falciparum. Materials and methods Blood samples of African patients with P. malariae (n 8) and P. ovale (n 9) malaria were used in order to modulate a robust shape equation for infected RBCs (IRBCs) from the early ring stage to the late schizont stage. Test specimens were prepared by centrifuging the patient’s blood at 1000 g for 5 min. After removal of the plasma, the RBCs were washed in RPMI 1640 and thin blood films were prepared by Giemsa staining. Blood samples from healthy donors (n 10) were used as controls. Finally, the thin blood films of the Pm-RBCs and Po-RBCs were imaged using an optical microscope (Olympus BX41, with Olysia software; Olympus Corporation, Tokyo, Japan) with 100/1.40 oil objective. Structural measurements and modeling The morphological characteristics of healthy and parasitized RBCs were obtained using a previously described method [24]. The final shape equation is represented by equation 1.
(
(Y b )2 X 2
ae
) ae((Y c )
2
X2
) d
(1)
Mathematical modeling resulted in the above shape equation, which consists of 4 constant coefficients a, b, c, and d. These constant parameters are obtained based on fitting of the mathematical model to the morphological characteristics including diameter (D) and 2 thicknesses (t1 and t2) of healthy RBCs (HRBCs) and IRBCs. Finally, 2-dimensional (2D) shape equations of RBCs at each stage of infection were drawn using Wolfram Mathematica version 8 (Wolfram Research, Champaign, IL, USA).
Figure 1. Diagram of a red blood cell showing its morphological characteristics.
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370 A. Karimi et al.
Figure 2. Optical micrographs of Giemsa-stained blood films as well as SPIP images of red blood cells infected by Plasmodium malariae, presenting the 3 stages of infection: ring (a, a1), trophozoite (b, b1), and schizont (c, c1).
Figure 3. Optical micrographs of Giemsa-stained blood films as well as SPIP images of red blood cells infected by Plasmodium ovale, presenting the 3 stages of infection: ring (a, a1), trophozoite (b, b1), and schizont (c, c1).
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Results and discussion The morphological characteristics measured were the diameter (D), thickness at the thickest part of the RBC (t1), and central thickness (t2), as shown in Figure 1. Optical micrographs of Giemsa-stained blood films as well as Scanning Probe Image Metrology A/S, Hørsholm, Denmark Processor (SPIP;) images of RBCs infected by P. malariae are shown in Figure 2. The figure shows the RBCs parasitized by P. malariae at the 3 stages of infection, including ring, trophozoite, and schizont. Figure 3 shows the same images for RBCs parasitized by P. ovale. The SPIP images representing the Z-range for both P. malariae and P. ovale infected RBCs obtained from thin blood smear are shown in Figure 4. The results showed that the Z-range of parasitized RBCs at the schizont stage increased 2.40 times for Pm-RBCs and 2.69 times for Po-RBCs compared to the HRBCs. Interestingly,
Figure 4. SPIP images plus Z-range of red blood cells infected by Plasmodium malariae (a) ring, (b) trophozoite, and (c) schizont, as well as Plasmodium ovale (d) ring, (e) trophozoite, and (f) schizont.
An innovative morphology-based diagnosis method 371 the diameter (D) of the Po-RBCs increased more significantly during parasite maturation (ring to schizont) compared to the Pm-RBCs (Figure 5), whereas the thicknesses (t1 and t2) increased similarly for both Pm-RBCs and Po-RBCs as the intraerythrocytic parasite grew. Table I shows the diameter (D), thicknesses (t1 and t2), and Z-range pixels of HRBCs, Pm-RBCs, and Po-RBCs. Using the parameters D, t1, and t2, a general 2D shape equation is fitted (equation 2). (
(Y b )2 X 2
ae
) ae((Y c )
2
) d ,(x range, y range)
X2 )
(2)
In order to obtain the constant coefficients a, b, c, and d for Pm-RBCs and Po-RBCs, the shape equations at each stage of infection were fitted to the parameters D, t1, and t2. Table II shows the constant parameters along with the related shape equations. Based on the shape equations it was possible to plot the shape of infected RBCs at each stage of infection. The results indicated that Pm-RBCs at the ring, trophozoite, and schizont stages had diameters of 7.89, 8.23, and 8.39 mm, respectively. Our previous study indicated that Pf-RBCs had diameters of 7.92, 8.28, and 8.47 mm at the ring, trophozoite, and schizont stages, respectively. The Po-RBCs showed diameters of 8.86, 9.86, and 10.96 mm, which are similar to those of Pv-RBCs at 8.90, 9.52, and 10.86 mm respectively, for ring, trophozoite, and schizont stages [24]. This is in agreement with previous studies, which have reported almost equal diameters for Pm-RBCs and Pf-RBCs, as well as Po-RBCs and Pv-RBCs. This could make the process of malaria diagnosis very difficult, which may lead to the wrong treatment procedure. From the ring to subsequent schizont stage, Pm-RBCs and Po-RBCs retained their biconcave shape (69% and 77% increase in t2 for Pm-RBCs and Po-RBCs, respectively, compared to the ring stage) (Table I, Figure 5). It is known that at the ring stage, a Pv-RBC retains its biconcave shape while a Pf-RBC almost loses its biconcave shape (50% and 93.40% increase in t2 for Pf-RBCs and Pv-RBCs, respectively, compared to ring stage) [24]. This is the reason why Pv-RBCs at the ring stage can still circulate freely in the peripheral blood [25]. Figure 6 presents the 2D shapes of Pf-RBCs, Pv-RBCs, Pm-RBCs, and Po-RBCs at the trophozoite stage, measured and plotted using the shape equations, in order to facilitate the morphological comparison between RBCs infected with the 4 different species of Plasmodium. The results of the present study showed that as the parasite enlarges, the Pm-RBC retains its biconcave shape, whilst the Pf-RBC almost entirely loses its biconcave shape and becomes nearly spherical at the
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372 A. Karimi et al.
Figure 5. 2D shapes of red blood cells parasitized by Plasmodium malariae (a) ring, (b) trophozoite, and (c) schizont, as well as Plasmodium ovale (d) ring, (e) trophozoite, and (f) schizont.
An innovative morphology-based diagnosis method 373
Table I. Diameter, thickness, and Z-depth of healthy and parasitized red blood cells measured by Olysia and SPIP software.
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Stage Plasmodium malariae Z-depth (pixel)a (number of cells)b D (mm)c t1 (mm)c t2 (mm)c Plasmodium ovale Z-depth (pixel)a (number of cells)b D (mm)c t1 (mm)c t2 (mm)c
HRBC
Ring
Trophozoite
Schizont
45.00 5.13 (40) 7.38 0.36 3.35 0.16 2.92 0.14
64.12 3.08 (37) 7.89 0.17 4.77 0.28 4.14 0.21
78.98 6.12 (34) 8.23 0.31 5.87 0.39 5.11 0.35
108.25 2.43 (32) 8.39 0.29 8.04 0.38 7.02 0.34
45.00 5.13 (40) 7.38 0.36 3.35 0.16 2.92 0.14
68.33 4.13 (35) 8.86 0.52 5.08 0.42 4.43 0.36
96.25 3.58 (33) 9.86 0.36 7.13 0.52 6.21 0.32
121.12 5.61 (30) 10.96 0.45 9.01 0.43 7.85 0.46
SPIP, Scanning Probe Image Processor; HRBC, healthy red blood cell. shows the depth of each red blood cell measured using SPIP software. bNumber of cells tested at each stage of parasite maturation inside the red blood cells. cData are reported at a significance level of p 0.05. aZ-depth
trophozoite and subsequent schizont stage (8.47, 9.19, and 8.01 mm for D, t1, and t2, respectively) [24]. The Pv-RBCs and Po-RBCs, furthermore, retain their biconcave shape throughout parasite development even though their volumes increase significantly (8.14 and 7.33 mm for Pv-RBCs and 9.01 and 7.85 mm for Po-RBCs for t1 and t2 at the schizont stage, respectively) (Figure 5). It has also been reported that erythrocytes infected with P. vivax are elliptical; this is similar to the results found in the present study for P. ovale, in which RBCs were found to have an elliptical and elongated shape [24,26]. The morphological characteristics of the IRBCs presented here could explain the significant increase in viscosity of the blood flow in the microcircular channel at the trophozoite stage, while at the ring stage, a similar viscosity to the healthy state model is
reported [27]. There have been substantial efforts to simulate the rheological behavior of Pf-RBCs in order to fundamentally understand the pathology of the disease and to predict the increase in flow resistance at each parasitemia level. However, in these studies, HRBCs and IRBCs were considered biconcave and spherical, respectively [19,27,28]. Cell geometry plays the main role in modeling and hemodynamic studies, and the shape of the RBC needs to be as accurate as possible. Therefore, it is believed that the shape equations introduced here could be used in rheological modeling studies to explain the rheological alterations caused by morphological changes in IRBCs. Since there has been no study on the rheological changes induced by Pm-RBCs or Po-RBCs, the shape information will be of benefit for such investigations. Most importantly we believe that the SPIP imaging method used
Table II. Two-dimensional shape equations for the healthy and infected red blood cells. Plasmodium malariae
(
) 30e((Y 2.35)
2
χ2
) 5101 , (6,6), (4.38, 3) ) (
(
) 30e((Y 2.10 )
2
χ2
) 6102 , (6,6), (4.60, 3.60) ) (
(Y 0.97 )2 χ2
HRBC
30e
Ring
30e
Trophozoite
50e
Schizont
80e
(Y 1.05 )2 χ2
(
) 50e((Y 1.90 )
χ2
) 15103 , (6,6), (4.80, 4) ) (
(
) 80e((Y 1.70 )
χ2
) 4105 , (6,6), (5.60, 5) ) (
(Y 1.1)2 χ2
(Y 1.1)2 χ2
2
2
Plasmodium ovale
(
(Y 0.97 )2 χ2
HRBC
30e
Ring
75e
Trophozoite
84e
Schizont
105e
HRBC, healthy red blood cell.
(
(Y 1)2 χ2
2
) 75e((Y 2.50 )
(
2
(Y 0.95 )2 χ2
(
) 30e((Y 2.35)
(Y 1)2 χ2
χ2
) 84e((Y 2.20 )
2
) 105e((Y 2 )
2
χ2
) 5101 , (6,6), (4.38, 3) ) (
) 8102 , (6,6), (5.20,3.70) ) (
χ2
χ2
) 6104 , (6,6), (5.70, 4.40) ) (
) 1105 , (6,6), (6.10,5.10) ) (
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374 A. Karimi et al.
Figure 6. 2D shapes of red blood cells parasitized by Plasmodium falciparum (a), Plasmodium vivax (b), Plasmodium malariae (c), and Plasmodium ovale (d), at the trophozoite stage.
in this study should be considered a new, easy, and additional method for the imaging of malaria parasites in order to quickly diagnose the type and stage of infection. In conclusion, the intention of this work was to introduce a new and simple method that has potential clinical use in the prediction, diagnosis, and treatment of human malaria for the clinicians, researchers, engineers, and technicians working on this disease. We believe that this method could be used to improve the accuracy of the Giemsa staining diagnosis method. We also hope that the results of this study will lead to a better understanding of the effect of morphological alterations on blood velocity and rheological changes induced by parasitized RBCs at each stage of infection. Acknowledgements This work was supported by the Iran University of Science and Technology and National Institute of
Genetic Engineering and Biotechnology. We gratefully acknowledge the participants for their cooperation and blood sample donation. We also thank the staff of the Mycology Department and Parasitology Laboratory of Tehran University of Medical Sciences for their collaboration. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Cisse B, Sokhna C, Boulanger D, Milet J, Bael H, Richardson K, et al. Seasonal intermittent preventive treatment with artesunate and sulfadoxine–pyrimethamine for prevention of malaria in Senegalese children: a randomised, placebo-controlled, double-blind trial. Lancet 2006;367: 659–67. [2] Williams TN. Red blood cell defects and malaria. Mol Biochem Parasitol 2006;149:121–7.
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[3] Chiyaka C, Mukandavire Z, Das P, Nyabadza F, Hove-Musekwa SD, Mwambi H. Theoretical analysis of mixed Plasmodium malariae and Plasmodium falciparum infections with partial cross-immunity. J Theor Biol 2010; 263:169–78. [4] Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. Vivax malaria: neglected and not benign. Am J Trop Med Hyg 2007;77:79–87. [5] Zakeri S, Raeisi A, Afsharpad M, Kakar Q, Ghasemi F, Atta H, et al. Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3alpha and pvcsp genes as molecular markers. Parasitol Int 2009;59:15–21. [6] Patarroyo MA, Perez-Leal O, Lopez Y, Cortes J, Rojas-Caraballo J, Gomez A, et al. Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2. Biochem Biophys Res Commun 2005; 337:853–9. [7] Fuehrer HP, Habler VE, Fally MA, Harl J, Starzengruber P, Swoboda P, et al. Plasmodium ovale in Bangladesh: genetic diversity and the first known evidence of the sympatric distribution of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in southern Asia. Int J Parasitol 2012; 42:693–9. [8] Guimarães LO, Bajay MM, Wunderlich G, Bueno MG, Röhe F, Catão-Dias JL, et al. The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil. Acta Trop 2012;124:27–32. [9] Mueller I, Zimmerman PA, Reeder JC. Plasmodium malariae and Plasmodium ovale—the ‘bashful’ malaria parasites. Trends Parasitol 2007;23:278–83. [10] Craig MH, Bredenkamp BL, Williams CH, Rossouw EJ, Kelly VJ, Kleinschmidt I, et al. Field and laboratory comparative evaluation of ten rapid malaria diagnostic tests. Trans R Soc Trop Med Hyg 2002;96: 258–65. [11] Ochola LB, Vounatsou P, Smith T, Mabaso ML, Newton C. The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis 2006;6:582–8. [12] Adeoye GO, Nga IC. Comparison of quantitative buffy coat technique (QBC) with Giemsa-stained thick film (GTF) for diagnosis of malaria. Parasitol Int 2007;56: 308–12. [13] Afzan MY, Sivanandam S, Kumar GS. Modified Field’s staining—a rapid stain for Trichomonas vaginalis. Diagn Microbiol Infect Dis 2010;68:159–62. [14] Clendennen TE III, Long GW, Baird JK. QBC® and Giemsastained thick blood films: diagnostic performance of laboratory technologists. Trans R Soc Trop Med Hyg 1995; 89:183–4.
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