Figure 2: Characterization of Drug Sensitivity, Size, Frequency, and Reactivation of Small Forms in MPCCs Match Hypnozoite Criteria.

(A) Primaquine (PQ) and atovaquone (AQ) were dosed at varying concentrations, starting 3 hours post infection (hpi) and replaced with daily media changes until day 5 when cultures were fixed (prophylactic mode). IC50 curves were produced by plotting fraction of EEFs remaining in culture at each concentration, as measured against untreated control wells. (mean±sem from triplicate wells, representative experiment shown, isolate VK210).

(B) Data from (a) was replotted, after segmenting the populations of parasites into ‘large’ and ‘small’ subpopulations, according to a 10μm diameter size threshold. Resulting curve fits show differential effect of PQ and AQ on small (<10 μm in diameter) and large (>10 μm in diameter) parasite populations.

(C) Cultures were dosed with PQ (5μM) or AQ (270nM) at 3 hpi until day 5. Cultures were maintained with daily media changes until day 21, when the sizes of any remaining parasites were assessed (n=3 wells per independent experiment. 3 experiments pooled for control, two experiments pooled for AQ, 1 experiment for PQ). See also Figures S3A and S4D.

(D) Representative hypnozoites and candidate reactivated schizonts in day 21 cultures, stained with a panel of antibodies, as indicated. Scale bar, 5μm. See also Figure S3C.

(E) Number of parasites remaining per well after treatment with PQ or AQ under the same treatment regimen as in (c) (mean±sem, n=3). See also Figure S4D.

(F) Size histograms of day 21 parasites to set a threshold for hypnozoite size. (3 independent experiments, triplicate wells per experiment).

(G) Hypnozoite diameters from day 8 and day 21 cultures (3 independent experiments, triplicate wells per experiment). *P=0.0004, two-tailed unpaired t-test with Welch’s correction.

(H) Hypnozoite frequency was evaluated in 6 separate experiments on day 8. CSP subtype of each experiment is indicated on the x-axis. (mean±sem shown from at least triplicate wells per experiment. Number of parasites interrogated in each experiment are as follows: 118,211,145,265,179,66,199) *Kruskall-Wallis test