Hyperspectral analysis to assess gametocytogenesis stage progression in malaria-infected human erythrocytes.

SIGNIFICANCE: Developments of anti-gametocyte drugs have been delayed due to insufficient understanding of gametocyte biology. We report a systematic workflow of data processing algorithms to quantify changes in the absorption spectrum and cell morphology of single malaria-infected erythrocytes. These changes may serve as biomarkers instrumental for the future development of antimalarial strategies, especially for anti-gametocyte drug design and testing. Image-based biomarkers may also be useful for nondestructive, label-free malaria detection and drug efficacy evaluation in resource-limited communities. AIM: We extend the application of hyperspectral microscopy to provide detailed insights into gametocyte stage progression through the quantitative analysis of absorbance spectra and cell morphology in malaria-infected erythrocytes. APPROACH: Malaria-infected erythrocytes at asexual and different gametocytogenesis stages were imaged through hyperspectral confocal microscopy. The preprocessing of the hyperspectral data cubes to transform them to color images and spectral angle mapper (SAM) analysis were first used to segment hemoglobin (Hb)- and hemozoin (Hz)-abundant areas within the host erythrocytes. Correlations between changes in cell morphology and increasing Hz-abundant areas of the infected erythrocytes were then examined to test their potential as optical biomarkers to determine the progression of infection, involving transitions from asexual to various gametocytogenesis stages. RESULTS: Following successful segmentation of Hb- and Hz-abundant areas in malaria-infected erythrocytes through SAM analysis, a modest correlation between the segmented Hz-abundant area and cell shape changes over time was observed. A significant increase in both the areal fraction of Hz and the ellipticity of the cell confirms that the Hz fraction change correlates with the progression of gametocytogenesis. CONCLUSIONS: Our workflow enables the quantification of changes in host cell morphology and the relative contents of Hb and Hz at various parasite growth stages. The quantified results exhibit a trend that both the segmented areal fraction of intracellular Hz and the ellipticity of the host cell increase as gametocytogenesis progresses, suggesting that these two metrics may serve as useful biomarkers to determine the stage of gametocytogenesis.