Related Experiment Video
Updated: Oct 19, 2025
Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors
Published on: May 12, 2022
A murine malaria protocol for characterizing transmission blocking benefits of antimalarial drug combinations
Yehenew A Ebstie1, Alain R Tenoh Guedoung1, Annette Habluetzel1,2
1School of Pharmacy, University of Camerino, Camerino, Italy.
Background:
Current efforts towards malaria elimination include the discovery of new transmission blocking (TB) drugs and identification of compounds suitable to replace primaquine, recommended as transmission blocking post treatment after artemisinin combination therapy (ACT). High through put screening of compound libraries has allowed to identify numerous compounds active in vitro against gametocytes and insect early sporogonic stages, but few studies have been performed to characterize TB compounds in vivo. Here we propose a double TB drug Direct Feeding Assay (2TB-DFA), suitable to assess the combined effects of TB compounds.
Materials And Methods:
Plasmodium berghei GFPcon (PbGFPcon), BALB/c mice and Anopheles stephensi mosquitoes were used. Artemisinin (ART) and artesunate (AS) served as examples of artemisinins, NeemAzal® (NA), as a known TB-product with sporontocidal activity. DFA experiments were performed to assess the appropriate time point of administration before mosquito feeding and estimate suitable sub-optimal doses of the three compounds that allow combination effects to be appreciated.
Results:
Suboptimal dosages, that reduce about 50% of oocyst development, were recorded with ART in the range of 16-30 mg/ kg, AS 14-28 mg/kg and NA 31-38mg/kg. Ten hours before mosquito feeding (corresponding to 3.5 days after mouse infection) was determined as a suitable time point for mouse treatment with ART and AS and 1 hour for post-treatment with NA. ART given at 35 mg/kg in combination with NA at 40 mg/kg reduced oocyst density by 94% and prevalence of infection by 59%. Similarly, the combination of ART at 25 mg/kg plus NA at 35 mg/kg decreased oocyst density by 95% and prevalence of infection by 34%. In the 2TB-DFA, conducted with AS (20 mg/kg) and NA (35 mg/kg) the combination treatment reduced oocyst density by 71% and did not affect prevalence of infection. Applying 'Highest Single Agent' analysis and considering as readout oocyst density and prevalence of infection, cooperative effects of the combination treatments, compared with the single compound treatments emerged.
Conclusion:
This study suggests the 2TB-DFA to be suitable for the profiling of new TB candidates that could substitute primaquine as a post-treatment to ACT courses.
