Kinetics of parasitaemia (asexual stages only, black dots) and gametocytaemia (stages 1–5; black squares) by microscopic observation, represented as a percentage of total erythrocytes.

<p>The number of asexual forms increased up to maximum parasitaemia on day 11. From that time onwards, a rapid decrease occurred leading to a parasitaemia (asexual stages) close to zero on day 17. Sexual forms were first detected on day 7, and gametocytaemia reached a peak of 1.69% on day 13. The gametocytes, initially in early forms, developed into mature stages reaching stages IV and V from day 15 to 20. After day 15, more than 70% of the gametocytes are in stage IV–V.</p

Comparison of the activity against mature gametocytes (IC₅₀) using either the ATP bioluminescence assay or the microscopical enumeration.

<p>Comparison of the activity against mature gametocytes (IC₅₀) using either the ATP bioluminescence assay or the microscopical enumeration.</p

Comparison of the dose-response curves of artesunate and methylene blue obtained with the ATP bioluminescence assay (black dots) or by microscopic counting (open circles).

<p>Each point represents mean values of 4 replicates ± SD.</p

ATP level per gametocyte number (R² = 0.99) counted with the luminometer after serial dilution.

<p>Gametocytes were counted using a Neubauer chamber. Each point represents mean values of 3 replicates ± SD.</p

The ability of key molecules to provoke growth retardation of HepG2 cells.

<p>Data were compared to the antimalarial potency of the same molecules against the development of <i>P. yoelii</i> liver schizonts following infection of HepG2 cells.</p><p>CI, cytotoxicity index.</p

The main classes of antimalarials.

<p>The chemical structures of all the main classes of antimalarials and other therapeutic and control molecules are assembled according to either the chemical classes they belong to (endoperoxides, 4- and 8- AQs, amino-alcohols) or their function (antifolate, antibiotics), or both (e.g., sulfonamides, a chemical class of antibiotic used in combined antimalarial therapies). The colour code associated with each class is consistent in all the figures in this report.</p

Validation and results of anti-infectives tested against <i>P. yoelii</i> liver stage parasites.

<p>(A) Opera-generated images from a time point experiment using a 20× objective lens. <i>P. yoelii</i> parasites (red) were visualized using a mouse polyclonal HSP70 antibody, and the HepG2 cells (green) were stained with Hoechst 33342 nucleic acid dye. Median parasite areas for the 15-h, 27-h, and 51-h time points were calculated as 16, 52, and 158 µm², respectively, using a custom Acapella script. (B) A dose response plot with atovaquone from data generated from the Opera imaging system. The median areas of compound-treated parasites were compared to untreated DMSO controls to determine the degree of inhibition. The IC₅₀ value of atovaquone is 22 nM. (C) From the Acumen ^eX3 imaging system, a 384-well heat map plate image of <i>P.yoelii</i> fluorescence intensities from a dose response experiment with the most active anti-infectives. The three wells at the top left corner were not infected with sporozoites and served as a negative control. Wells were treated with a 1∶3 dilution of compounds starting at 30 µM (0.1% DMSO). In the experiments where the highest compound concentration tested was 10 µM, the entry is labelled 10. Compounds used are: naphthoquine (naph.), pyrimethamine (pyri.), trimethoprim (trim.), deferoxamine (defe.), artemisone (artem.), atovaquone (ato.), artesunate (artes.), cycloguanil (cyclo.), methylene blue (met. bl.), quinidine (quin.), dihydroartemisinin (DHA). Triangles represent the dilution steps of the drugs as described above. Stars designate the three wells with no sporozoite. (D) IC₅₀ results from a compound dose response experiment performed in a 384-well plate format. Results were generated from the Opera using median parasite area to determine level of inhibition.</p

The transmission-blocking potential of selected antimalarials in three bioassays that cover different phases of <i>Plasmodium</i> vector stage development.

<p>(A) Assays examined exflagellation (<i>P. falciparum</i>), ookinete formation (<i>P. berghei</i>), or oocyst formation (<i>P. falciparum</i>). All antimalarials were screened at 10 µM. (B) The biological content of the in vitro <i>P. berghei</i> ookinete assay spans gamete formation, fertilization, zygote development, and ookinete formation. Ookinete formation was insensitive to most of the antimalarials tested. Atovaquone, cycloheximide, pyronaridine, pyrimethamine, and thiostrepton all strongly inhibited ookinete formation (<i>p</i><0.03 by Student's <i>t</i>-test), while tafenoquine, cis-mirincamycin, and fosmidomycin gave an enhancement of ookinete formation that was not statistically significant. (C) The medium throughput fluorescent ookinete assay determined IC₅₀ values for pyronaridine, thiostrepton, cycloheximide, and atovaquone of 6 µM, 8 µM, 25 nM, and 65 nM, respectively. (D) The in vitro <i>P. falciparum</i> exflagellation assay exposes mature gametocytes to antimalarials for 24 h before triggering exflagellation. 16 out of 29 antimalarials tested, including all but one of the endoperoxides and 4-AQs, showed a statistically significant >50% inhibition of exflagellation (<i>p</i><0.05). Pyronaridine, tert-butyl isoquine, NPC-1161B, OZ277, and cycloheximide all inhibited exflagellation totally at 10 µM. All experiments in triplicate. (E) The in vivo <i>P. falciparum</i> oocyst assay differs from the <i>P. berghei</i> oocyst assay in that mature gametocytes were exposed to the antimalarials in culture for 24 h before feeding to mosquitoes. The endoperoxides all strongly reduced transmission. NPC1161-B, lumefantrine, halofantrine, and mefloquine +RS isomer were also active. (<i>n</i> = 4–61 observations; average ± standard error of the mean [SEM]).</p

Summary of the activity of the most widely used antimalarials throughout the life cycle of <i>Plasmodium</i>.

<p>The three main phases, i.e., liver stage, blood stage, and vector stage, of the life cycle of <i>Plasmodium</i> are shown. The two key entry points leading to transmission of the parasites from vector to host and from host to vector are indicated (green circles). Parasite forms specific to each stage are highlighted and drugs identified as inhibitors of development of these forms are listed in boxes and coloured as described in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001169#pmed-1001169-g001" target="_blank">Figure 1</a>. Stars highlight components of the main artemisinin combination therapies: green, coartem; red, pyramax; orange, eurartesim; blue, ASAQ.</p

Concept of PNSA in vivo assays.

<p>Comparison of the theoretical growth curves of <i>Plasmodium berghei</i> upon intravenous infection at day 0 under (A) a Peters’ 4-day test-type or (B) the PNSA assay format for the evaluation of the antimalarial efficacy of drugs. The solid curves represent the growth of parasites treated with vehicle. The dotted lines represent the growth of parasites under arbitrary treatments (×n, denotes arbitrary number of drug dosages) leading to ED₅₀, ED₉₀ and ED₉₉, respectively. The parasitemia that marks the limit between net growth and net clearance of the parasite circulating in peripheral blood is denoted as the NG line. The limit of quantification of parasitemia is denoted as the LQ line. PRR is the parasite reduction ratio, i.e. the ratio of the baseline parasite count to that following treatment.</p