BackgroundThe threat of malaria is still present in the world. Recognizing the type of parasite is important in determining a treatment plan. The golden routine involves microscopic diagnostics of Giemsa-stained thin blood smears, however, alternative methods are also constantly being sought, in order to gain an additional insight into the course of the disease. Spectroscopic methods, e.g., Raman spectroscopy, are becoming increasingly popular, due to the non-destructive nature of these techniques.MethodsThe study included patients hospitalized for malaria caused byPlasmodium falciparumorPlasmodium vivax,in the Department of Infectious Diseases at the University Hospital in Krakow, Poland, as well as healthy volunteers. The aim of this study was to assess the possibility of using Raman spectroscopy and 2D correlation (2D-COS) spectroscopy in understanding the structural changes in erythrocytes depending on the type of attacking parasite. EPR spectroscopy and two-trace two-dimensional (2T2D) correlation was also used to examine the specificity of paramagnetic centres found in the infected human blood.ResultsTwo-dimensional (2D) correlation spectroscopy facilitates the identification of the hidden relationship, allowing for the discrimination of Raman spectra obtained during the course of disease in human red blood cells, infected byP. falciparumorP. vivax. Synchronous cross-peaks indicate the processes taking place inside the erythrocyte during the export of the parasite protein towards the cell membrane. In contrast, moieties that generate asynchronous 2D cross-peaks are characteristic of the respective ligand-receptor domains. These changes observed during the course of the infection, have different dynamics forP. falciparumandP. vivax, as indicated by the asynchronous correlation cross-peaks. Two-trace two-dimensional (2T2D) spectroscopy, applied to EPR spectra of blood at the beginning of the infection, showed differences betweenP. falciparumandP. vivax.ConclusionsA unique feature of 2D-COS is the ability to discriminate the collected Raman and EPR spectra. The changes observed during the course of a malaria infection have different dynamics forP. falciparumandP. vivax, indicated by the reverse sequence of events. For each type of parasite, a specific recycling process for iron was observed in the infected blood.Graphical Abstract