The ring-stage of Plasmodium falciparum observed in RBCs of hospitalized malaria patients

Abstract

Raman spectra of the blood samples obtained directly from hospitalized malaria patients with Plasmodium falciparum (P. falciparum) in the ring-stage were analyzed. Changes observed in the Raman band intensities of the infected patients compared to healthy volunteers are the result of parasite activity inside red blood cells. The obtained spectra were discussed by analyzing differences in particular spectral regions by evaluating changes in the band intensity ratios as well as using PCA analysis. The alterations of erythrocyte membranes caused by parasite penetration are visible by a reduced I₁₁₃₀/I₁₀₇₅ intensity ratio expressing the lowering of the amount of domains arranged in trans conformation. The I₂₉₃₀/I₂₈₅₀ ratio, which is a measure of modifications in structures of membrane proteins and lipids, in infected red blood cells increases, which is caused by malaria protein export to the erythrocyte membrane and expresses the membrane disarrangement. In the pyrrole ring vibration region, the ν₄ band marker of the oxygenated-Hb shows at 1371 cm⁻¹ whereas the ν₄ band at 1353 cm⁻¹ related to the deoxygenated-Hb is observed for malaria patients and is characterized by a higher intensity in infected erythrocytes. The amide I analysis shows the modifications in the secondary structure composition in the infected RBCs. We found that the P. falciparum infection leads to a decrease in the α-helical content and a concurrent increase in undefined (random-coil) structures. It was observed that the Raman spectra changes are also the result of the hemozoin formation process. In the pyrrole ring stretching vibration region, the increase of 1220 cm⁻¹ (deoxyHb) as against 1248 cm⁻¹ (oxyHb) may be considered as a signal of hemozoin formation in the RBCs. Relatively intense band patterns at 1560 cm⁻¹ and also at 1570 cm⁻¹ and 1552 cm⁻¹ may be due to the hemozoin that is formed according to parasite activity. The results of medical diagnostic tests had not presented changes in patient RBC parameters. A significant reduction in WBC count was noticed along with a decrease in neutrophil and platelet count when compared with the control group. Although no change is observed in the overall picture of the erythrocytes, pathological changes are evident in the Raman spectrum.