Shape of RBCs affects parasite proliferation in SS cells. (A) AA RBCs infected with B divergens were imaged at day 3 of infection. More than 99% of the cells were classified as “round” by the ML algorithm as shown in the representative images. Cells were stained with anti-GPA (violet), Vybrant Green (nucleus of Babesia), and anti–HbF-APC. All cells expressed GPA, and Babesia was seen as green dots in the cells with 1N, 2N, and 4N nuclei. (B) AS cells were similarly stained and analyzed by image cytometry. More than 98% of the cells were classified as “round” by the ML algorithm, and Babesia was seen as green dots in the cells with 1N, 2N, and 4N nuclei. (C-D) SS cells were stained similarly and imaged at low O₂ (C) and high O₂ (D) conditions. A fraction of cells appeared as sickled at both concentrations, although the “sickle” population was significantly higher at low O₂ conditions, as shown in the images. Most SS cells that were classified as “sickle” did not harbor the parasite, and for the few which did, the DNA content was usually always found to be 1N. The “round” cells classified in SS RBCs were shown to be the predominant cell harboring the parasite. (E) The percentage of cells expressing HbF was plotted (mean ± standard error of the mean) for AA (1.843 ± 0.4087), AS (4.755 ± 1.123), and SS (51.17 ± 1.123). The difference in HbF⁺ cells was statistically significant among the genotypes. (F) The percentage of cells that were classified as “sickle” by the ML algorithm was plotted at different O₂ levels for Babesia-infected SS cells (n = 5). The percentage of sickling cells reduced as the cells were incubated at higher O₂ levels, and this change was statistically significant (P = .0357). (G) Babesia-infected cells were gated and then checked for “sickle” or “round” shape in infected SS samples. As evident from the graph, almost all Babesia-infected host SS cells were “round” and not “sickle” with a P value of <.0001. Differences in these were not significant between different O₂ concentrations; therefore, across all different O₂ concentrations tested, Babesia invaded “round” cells more often than “sickle” cells. (H) The proportion of Babesia⁺ cells in normalized HbF⁺ or normalized HbF^–- cells grown under 1% O₂ was calculated. Thus, if HbF⁺ and HbF^– cells are present in similar numbers, parasites prefer HbF⁺ cells over HbF^– cells (P = .0055, n = 8). (I) Comparison of the percentage of all parasitized cells that are HbF⁺ at 1% and 21% O₂. As compared with parasites at 1% O₂, parasites at 21% O₂ seem to have a diminished preference for HbF⁺ cells (P = .0047, n = 10). Scale bar: Enlarged Brightfield (BF) image shown beside Fig. 4C.; scale = 7 μm.