Use of Plasmodium falciparum culture-adapted field isolates for in vitro exflagellation-blocking assay

Abstract

BackgroundA major requirement for malaria elimination is the development of transmission-blocking interventions. In vitro transmission-blocking bioassays currently mostly rely on the use of very few Plasmodium falciparum reference laboratory strains isolated decades ago. To fill a piece of the gap between laboratory experimental models and natural systems, the purpose of this work was to determine if culture-adapted field isolates of P. falciparum are suitable for in vitro transmission-blocking bioassays targeting functional maturity of male gametocytes: exflagellation.MethodsPlasmodium falciparum isolates were adapted to in vitro culture before being used for in vitro gametocyte production. Maturation was assessed by microscopic observation of gametocyte morphology over time of culture and the functional viability of male gametocytes was assessed by microscopic counting of exflagellating gametocytes. Suitability for in vitro exflagellation-blocking bioassays was determined using dihydroartemisinin and methylene blue.ResultsIn vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia. Functional maturity of male gametocytes was assessed by exflagellation observations and all four isolates could be used in exflagellation-blocking bioassays with adequate response to methylene blue and dihydroartemisinin.ConclusionThis work shows that in vitro culture-adapted P. falciparum field isolates of different genetic background, from South America and Southeast Asia, can successfully be used for bioassays targeting the male gametocyte to gamete transition, exflagellation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0752-x) contains supplementary material, which is available to authorized users.

Methylene blue induced morphological deformations in Plasmodium falciparum gametocytes: implications for transmission-blocking

“…Moreover, gametocyte production potential in RKL-9 and JDP-8 appeared to be stable after multiple freeze–thaw cycles. Other studies also reported no loss in gametocyte production upon maintenance of isolates in asexual culture for 18 months [ 22 ] and also after cryopreservation [ 13 , 26 , 27 ]. In vitro gametocyte production potential of parasite is strain specific [ 28 ], exhibited in response to ‘nonspecific’ stress in the form of environmental stimuli [ 22 , 23 , 29 ].…”

“…This might be because very few isolates can reproducibly generate gametocytes in high yield in vitro and also show a gradual loss of gametocyte producing potential in continuous culture [ 23 ]. This makes it difficult to study the process of gametocytogenesis [ 23 , 24 ] and to perform gametocytocidal drug screening due to the dependency on cryopreserved stabilates with minimum passage in order to preserve gametocyte production phenotype [ 13 ]. The two field isolates, RKL-9 and JDP-8 used in present study did not show any significant reduction in gametocyte production potential in vitro for at least 6 months in asexual culture.…”

“…Toxicity of methylene blue is dose and intrinsic G6PD activity dependent; similar to primaquine but with a difference that methylene blue exerts its oxidizing (haemolytic) properties at higher doses [ 6 , 7 ]. In vitro and ex vivo studies performed with methylene blue against laboratory reference strains reported its late stage gametocytocidal activity [ 8 – 13 ]. Although a handful of reference strains such as 3D7 are useful to standardize and validate high throughput assays, a more realistic representation of drug efficacy is derived when potency of drug is evaluated on parasites that have been selected after years of multiple drug pressure and natural transmission.…”

“…Although a handful of reference strains such as 3D7 are useful to standardize and validate high throughput assays, a more realistic representation of drug efficacy is derived when potency of drug is evaluated on parasites that have been selected after years of multiple drug pressure and natural transmission. Evaluation of drug efficacy against gametocytes produced from these field isolates is of utmost importance as anti-malarial drugs respond differently to them as compared to culture adapted reference strains [ 13 ].…”

Antiplasmodial activities of dyes against Plasmodium falciparum asexual and sexual stages: Contrasted uptakes of triarylmethanes Brilliant green, Green S (E142), and Patent Blue V (E131) by erythrocytes

“…Exflagellation inhibition assays were performed using published protocols ( Leba et al., 2015 , Bhattacharyya and Kumar, 2001 ). Briefly, 1.5 mL tube containing 170 μL of culture medium (RPMI 1640 medium with 25 mM HEPES, 50 mg/L hypoxanthine, 2 g/L sodium bicarbonate, 10% human serum) was preheated at 37 °C with the dye pre-dissolved in water for a final concentration of 1 μM.…”