Fig. 3: Osmotic stability of P. vivax-infected reticulocytes.

a Flow cytometry strategy for measuring osmotic stability Plasmodium-infected RBCs. Created with BioRender.com. b Representative lysis curves for P. vivax-infected CD71+ (DNA+ CD71+, blue line) and CD71− reticulocytes (DNA+ CD71−, red line) and uninfected CD71+ reticulocytes (DNA− CD71+, gray line) and normocytes (DNA− CD71−, purple line) from a cryopreserved Brazilian clinical P. vivax sample after 1-h in vitro culture. Data fit with least-squares regression fit curves of normalized data. c Developmental stage of in vitro cultured ex vivo Indian (n = 2) and cryopreserved Brazilian (n = 5) P. vivax clinical isolates. Horizontal lines and error bars represent mean ± SEM. Inset are representative images of P. vivax IDC development stages. Scale bar, 10 μm. Gray (stage I), green (stage II), blue (stage III), red (stage IV), yellow (stage V), purple (gametocyte) bars indicate IDC development stage. d P. vivax-infected reticulocytes from cryopreserved Brazilian (black symbols) and non-cryopreserved Indian (blue symbols) clinical P. vivax samples lysis₅₀ values after 1-, 8-, 16-, 24-, and 44-h of in vitro culture. Unique symbols indicate biological replicates. Horizontal lines and error bars represent mean ± SEM. Dashed line (154 mOsm) and gray shading indicate lysis₅₀ threshold for hemolytic anemias. Hashtag and n.s., significant and no significant difference in cryopreserved Brazilian and non-cryopreserved Indian P. vivax lysis₅₀ values, respectively (1-h p = 0.05, 8-h p = 0.6, 24-h p = 0.8, 44-h p = 1.0) using unpaired two-tailed Mann–Whitney test. Astericks and n.s., significant and no significant difference between Brazilian P. vivax-infected reticulocytes lysis₅₀ at 24- and 1-, 8-, 16-, 24- and 44-h of culture, respectively (1-h p = 0.3, 8-h p = 0.02, 16-h p = 0.04, and 44-h p = 0.9) using unpaired two-tailed Dunnett’s multiple comparisons test. e Cryopreserved Brazilian P. vivax-infected CD71+ and CD71− lysis₅₀ values normalized to lysis₅₀ values of uninfected CD71+ reticulocytes at 8- (n = 3), 16- (n = 5), and 24-h (n = 5) of culture. Unique symbols indicate biological replicates. Horizontal lines and error bars represent mean ± SEM. Astericks and n.s., significant and no significant difference between 24-h and 8- and 16-h of culture lysis₅₀ values, respectively (CD71+: 8-h p = 0.04, 16-h p = 0.006 and CD71-: 8-h p = 0.1, 16-h p = 0.05) using unpaired two-tailed Student’s t-test.