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

Leba LJ, Musset L, Pelleau S, Estevez Y, Birer C, Briolant S, Witkowski B, Ménard D, Delves MJ, Legrand E, Duplais C, Popovici J - Malar. J. (2015)

Bottom Line: Plasmodium 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.In vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Parasitologie, Institut Pasteur de la Guyane, Cayenne, French Guiana, France. louisjerome973@hotmail.fr.

ABSTRACT

Background: A 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.

Methods: Plasmodium 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.

Results: In 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.

Conclusion: This 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.

No MeSH data available.


Related in: MedlinePlus

Exflagellation levels over days of gametocyte culture (arbitrary unit). For each independent culture, exflagellation centres values are normalized to the highest value of the culture (data are presented in Additional file 1). The mean and SEM of three cultures is shown for 7G8 and the two French Guiana strains (Q188 and Q206) while a single culture for each Cambodian isolate is represented (6831 and 6836)
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Fig1: Exflagellation levels over days of gametocyte culture (arbitrary unit). For each independent culture, exflagellation centres values are normalized to the highest value of the culture (data are presented in Additional file 1). The mean and SEM of three cultures is shown for 7G8 and the two French Guiana strains (Q188 and Q206) while a single culture for each Cambodian isolate is represented (6831 and 6836)

Mentions: The protocols were first optimized by using the South American isolates prior to being verified with the Southeast Asian isolates. Gametocyte development was straightforward at the first attempt. Initially however, although stage V gametocytes were clearly identified in the cultures, no exflagellation was observed. This suggests that bioassays with a read-out based only on morphological development of gametocytes do not report on their onward functional viability, therefore they may be underpowered. As already observed by others [2, 4], the age of uninfected RBC when initiating the gametocyte culture was a critical factor for successful maturation: RBC stored at 4 °C more than ~ two to 3 days rapidly compromise maturation. Additionally, variability in human serum was found also to be a critical point for gametocyte maturation, so pools from different donors of serum were used when possible. Using fresh uninfected RBC, fully mature gametocytes capable of exflagellation were generated for all isolates. For all isolates and 7G8, gametocyte development time was similar and maturity peaked at ~16–18 days of culture, slightly longer than what is usually reported for 3D7, ~12–14 days (Fig. 1) [2, 11].Fig. 1


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

Leba LJ, Musset L, Pelleau S, Estevez Y, Birer C, Briolant S, Witkowski B, Ménard D, Delves MJ, Legrand E, Duplais C, Popovici J - Malar. J. (2015)

Exflagellation levels over days of gametocyte culture (arbitrary unit). For each independent culture, exflagellation centres values are normalized to the highest value of the culture (data are presented in Additional file 1). The mean and SEM of three cultures is shown for 7G8 and the two French Guiana strains (Q188 and Q206) while a single culture for each Cambodian isolate is represented (6831 and 6836)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4464717&req=5

Fig1: Exflagellation levels over days of gametocyte culture (arbitrary unit). For each independent culture, exflagellation centres values are normalized to the highest value of the culture (data are presented in Additional file 1). The mean and SEM of three cultures is shown for 7G8 and the two French Guiana strains (Q188 and Q206) while a single culture for each Cambodian isolate is represented (6831 and 6836)
Mentions: The protocols were first optimized by using the South American isolates prior to being verified with the Southeast Asian isolates. Gametocyte development was straightforward at the first attempt. Initially however, although stage V gametocytes were clearly identified in the cultures, no exflagellation was observed. This suggests that bioassays with a read-out based only on morphological development of gametocytes do not report on their onward functional viability, therefore they may be underpowered. As already observed by others [2, 4], the age of uninfected RBC when initiating the gametocyte culture was a critical factor for successful maturation: RBC stored at 4 °C more than ~ two to 3 days rapidly compromise maturation. Additionally, variability in human serum was found also to be a critical point for gametocyte maturation, so pools from different donors of serum were used when possible. Using fresh uninfected RBC, fully mature gametocytes capable of exflagellation were generated for all isolates. For all isolates and 7G8, gametocyte development time was similar and maturity peaked at ~16–18 days of culture, slightly longer than what is usually reported for 3D7, ~12–14 days (Fig. 1) [2, 11].Fig. 1

Bottom Line: Plasmodium 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.In vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Parasitologie, Institut Pasteur de la Guyane, Cayenne, French Guiana, France. louisjerome973@hotmail.fr.

ABSTRACT

Background: A 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.

Methods: Plasmodium 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.

Results: In 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.

Conclusion: This 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.

No MeSH data available.


Related in: MedlinePlus