Abstract
BackgroundMalaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening.MethodsPlasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination.ResultsPlasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC50 (early stages) as 424.1 nM and mean IC50 (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations.ConclusionsField isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field isolates and is proposed to be used as a gametocytocidal adjunct with artemisinin-based combination therapy. Further exploration of methylene blue in clinical studies amongst Indian population, including G6PD deficient patients, is recommended.
Highlights
Malaria remains a global health problem despite availability of effective tools
RKL-9 and JDP-8 collected from Rourkela and Jagdalpur, respectively, exhibited higher gametocytaemia (> 2%) (Table 1) than other isolates in vitro and were deemed most suitable for stage specific drug screening experiments
Gametocytes produced from isolates RKL-9 and JDP-8 followed a very reproducible progression of gametocyte maturation from stage I to stage V
Summary
Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose6-phosphate dehydrogenase (G6PD) deficient patients. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening. To achieve the dream of malaria elimination, preventing transmission is crucial and approaches targeting gametocytes are highly essential. Transmission-blocking drugs that can effectively target mature gametocytes are very few, out of which only primaquine is licensed for clinical use [1]. Associated safety risks in glucose-6-phosphate dehydrogenase (G6PD) deficient patients have limited its usage on a large scale [2]. Research efforts should be directed towards developing new transmission-blocking drugs that are safe for G6PD deficient patients
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