Discriminating the Intraerythrocytic Lifecycle Stages of the Malaria Parasite Using Synchrotron FT-IR Microspectroscopy and an Artificial Neural Network

Abstract

Synchrotron Fourier transform infrared (FT-IR) spectra of fixed single erythrocytes infected with Plasmodium falciparum at different stages of the intraerythrocytic cycle are presented for the first time. Bands assigned to the hemozoin moiety at 1712, 1664, and 1209 cm(-1) are observed in FT-IR difference spectra between uninfected erythrocytes and infected trophozoites. These bands are also found to be important contributors in separating the trophozoite spectra from the uninfected cell spectra in principal components analysis. All stages of the intraerythrocytic lifecycle of the malarial parasite, including the ring and schizont stage, can be differentiated by visual inspection of the C-H stretching region (3100-2800 cm(-1)) and by using principal components analysis. Bands at 2922, 2852, and 1738 cm(-1) assigned to the nu(asym)(CH(2) acyl chain lipids), nu(sym)(CH(2) acyl chain lipids), and the ester carbonyl band, respectively, increase as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage. Training of an artificial neural network showed that excellent automated spectroscopic discrimination between P. falciparum-infected cells and the control cells is possible. FT-IR difference spectra indicate a change in the production of unsaturated fatty acids as the parasite matures. The ring stage spectrum shows bands associated with cis unsaturated fatty acids. The schizont stage spectrum displays no evidence of cis bands and suggests an increase in saturated fatty acids. These results demonstrate that different phases of the P. falciparum intraerthyrocytic life cycle are characterized by different lipid compositions giving rise to distinct spectral profiles in the C-H stretching region. This insight paves the way for an automated infrared-based technology capable of diagnosing malaria at all intraerythrocytic stages of the parasite's life cycle.

Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria

“…Previous in vitro photoacoustic approaches (26,27) required a relatively large amount of blood because they used the bulk thermoelastic mechanism of signal generation and thus were unable to detect single iRBCs among many uninfected RBCs. Several other diagnostic methods use the various optical properties of hemozoin (19,(28)(29)(30)(31)(32)(33). All these methods cannot transdermally and rapidly detect low levels of parasitemia in a needle-and reagent-free way, as H-VNBs demonstrated.…”

“…Hemozoin can be found in any parasite type and any blood stage, including gametocytes (8,10,11). High parasite sensitivity and the specificity of H-VNBs result from a much stronger acoustic signal of a vapor nanobubble compared with that from a bulk thermal effect used by traditional photoacoustics (26,27,34) or from the optical effects in hemozoin (28)(29)(30)(31)(32)(33). Vapor bubbles can also be generated via optical absorbance of hemoglobin in uninfected RBCs but at more than 100-fold higher laser fluence (35).…”

Rapid transdermal bloodless and reagent-free malaria detection

“…Previous in vitro photoacoustic approaches 28,29 required relatively large amount of blood because they employed the bulk thermoelastic mechanism of signal generation and thus were unable to detect single iRBCs among many uninfected RBCs. Several other diagnostic methods use the various optical properties of hemozoin [30][31][32][33][34][35][36] . All these methods cannot transdermally and rapidly detect low levels of parasitemia in the needle-and reagent-free way as H-VNBs demonstrated.…”

Synchrotron Radiation Spectroscopic Techniques as Tools for the Medicinal Chemist: Microprobe X-Ray Fluorescence Imaging, X-Ray Absorption Spectroscopy, and Infrared Microspectroscopy

“…[92] There have since been significant developments towards disease diagnosis, improvements in the understanding of disease progression and validations of hypotheses regarding diseases such as prostate, [93] skin, [94] colon, [95] cervical, [94,96] and breast cancers, [97] melanoma, [98] leukemia, [99] transmissible spongiform encephalopathies/prion diseases, [100] Alzheimers disease, [101][102] diabetes, [103] malaria, [104] ophthalmic disorders, [105] and arthritic disorders. [94,99] The usefulness of SR-FTIR microspectroscopy derives from the fact that the IR source is 10-1000 times more brilliant than the conventional globar source.…”