A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes

Abstract

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 μM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 μM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

Keywords: culture; direct membrane feeding assay; drug assay; ex vivo; field-isolated gametocytes; transmission-blocking activity.

FIG 1

Infectivity of field-isolated gametocytes to Anopheles coluzzii at baseline and following ex vivo maintenance under culture conditions in various media for 24 h and 48 h. (A) Oocyst prevalence (number of positive mosquitoes) at baseline (WB and SR) and after a 24-h ex vivo exposure under culture condition in SCM, ACM, and SACM. At baseline, the mean oocyst prevalence was 30.6% and 38.2% for WB-DMFA and SR-DMFA, respectively. Compared with SR-DMFA, the mean oocyst prevalence significantly increased to 52.4% when collected gametocytes were maintained in SCM (P = 0.03). (B) Oocyst intensity (number of oocysts per mosquitoes) at baseline and after a 24-h ex vivo exposure under culture condition in SCM, ACM, and SACM. The mean oocyst intensity was significantly greater in SR-DMFA than that in WB-DMFA (5.02 versus 2.5 oocysts/mosquito, P = 0.02), suggesting that the removal of patient serum improved oocyst formation. (C) Oocyst prevalence at baseline and after ex vivo exposure under culture condition in SCM for 48 h. Oocyst prevalence from a sample maintained under culture condition is comparable to that of SR samples (23.9 versus 15.5, P = 0.1). (D) Oocyst intensity at baseline and after ex vivo exposure under culture condition in SCM for 48 h. Oocyst intensity from a sample maintained under culture condition is comparable to that of SR samples (0.6 versus 1.5 oocyst per mosquito P = 0.06). (E) Oocyst prevalence at baseline and after ex vivo exposure under culture condition in DMSO for 48 h. Infectivity was significantly higher when the mosquitoes were fed with SR samples than that when mosquitoes were fed with WB samples (43.9 versus 27.4; P < 10⁻⁴). Oocyst prevalence from samples maintained in DMSO under culture condition is comparable to that of SR samples (48.6 versus 43.9, P = 0.3). (F) Oocyst intensity at baseline and after ex vivo exposure under culture condition in DMSO for 48 h. Oocyst intensity was significantly higher in samples from SR-DMFA than that in samples from WB-DMFA (13 versus 8 oocyst per mosquito; P < 10⁻⁴). Oocyst intensity from samples maintained in DMSO under culture condition is comparable to that of SR samples (9.6 versus 13 oocyst per mosquito; P = 0.6). Plots represent the mean and the horizontal lines within the histogram indicate the standard deviation. Asterisks indicate significant (P < 0.05) differences between groups compared by the Wilcoxon signed-rank test. SCM, serum culture medium; ACM, AlbuMax culture medium; SACM, serum-AlbuMax culture medium; WB, whole blood; SR, serum replacement; DMFA, direct membrane feeding assay.

FIG 2

Evaluation of the activity of reference drugs on mosquito infectivity using the ex vivo transmission blocking assay. The histograms show the mean (± standard deviation) oocyst prevalence for the five experimental concentrations evaluated for each drug. (A) Chloroquine was ineffective to eliminate oocyst formation at 10 μM. (B) Dihydroartemisinin does not eliminate oocyst formation at 1 μM but has a transmission-blocking activity of ~78.5% at this concentration. (C) Primaquine inhibits oocyst formation by 19% at 1 μM and abolishes oocyst formation at 7.5 μM. (D) KDU691 prevented oocyst formation at 1 μM. (E) GNF179 shows high inhibition of gametocyte sexual life cycle progression in the mosquito as it abolished oocyst formation at 5 nM. (F) Oryzalin impairs oocyst formation at 1 μM with an oocyst reduction of 50%.

FIG 3

Dose response against oocyst formation. (A) Primaquine exhibited a half maximal inhibitory concentration (IC₅₀) of 1.3 μM and a full maximal inhibitory concentration (IC₁₀₀) of ~3.7 μM. (B) KDU691 exhibits an IC₅₀ of 110 nM and an IC₁₀₀ of ~200 nM. Oocyst prevalence was normalized using Prism transformation tools. First the oocyst prevalence from the DMSO control was set as 100%, and those of the various concentrations were transformed accordingly. For the plot, the three-parameter nonlinear regression model with the least-squares method to find the best fit was applied.

FIG 4

Experimental design flow chart for the ex vivo assessment of field-isolated gametocyte viability and infectivity. First, the baseline infectivity of field-isolated gametocytes is determined with the heparinized blood samples. The samples are treated and fed to the mosquito at 45 to 90 min after blood collection. Blood samples collected into acid citrate dextrose (ACD) anticoagulant tubes are diluted into specific culture media (serum culture medium [SCM], AlbuMax culture medium [ACM], and a serum-AlbuMax culture medium [SACM], DMSO control medium, drug-containing medium). The resulting samples are incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 of 48 h. Samples are subsequently prepared for direct membrane feeding assays (DMFAs). For DMFAs, infectious blood samples are fed to Anopheles coluzzii mosquitoes, and the presence (oocyst prevalence) and number of oocysts (oocyst intensity) in the gut of the mosquitoes are assessed 8 days after the infectious blood meal.