Use of Plasmodium falciparum culture-adapted field isolates for in vitro exflagellation-blocking assay
- Louis-Jérôme Leba1, 2,
- Lise Musset1,
- Stéphane Pelleau1,
- Yannick Estevez2,
- Caroline Birer2,
- Sébastien Briolant1, 3, 4,
- Benoit Witkowski5,
- Didier Ménard5,
- Michael J Delves6,
- Eric Legrand1,
- Christophe Duplais†2 and
- Jean Popovici†1, 5Email author
© Leba et al. 2015
Received: 14 April 2015
Accepted: 27 May 2015
Published: 4 June 2015
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.
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.
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.
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.
Among the actions required for malaria elimination, blocking the transmission of Plasmodium parasites from human to mosquitoes is critical . Passage through the vector is an obligatory step for the parasite to continue its life cycle and it relies exclusively on the most mature forms of the sexual stages, stage V gametocytes. Mosquito feeding assays remain the gold standard to evaluate transmission-blocking strategies but require resource-intensive techniques. To circumvent these technical difficulties, several in vitro transmission-blocking bioassays targeting the sexual stages of the parasite have been described with different endpoints and various interpretative values [2, 3]. Although the outputs of these in vitro assays are difficult to transpose in vivo, the most clinically relevant assay should be able to evaluate the ability of a compound to either kill stage V gametocytes (viability assay) or inhibit their ability to differentiate into later mosquito stages (functional maturity assay). It is possible to induce in vitro gamete formation from Plasmodium falciparum male and female gametocytes where male gametocytes undergo major differentiation leading to the production of eight mobile gametes, by a process called exflagellation, which is easily observed visually.
Currently, the majority of P. falciparum in vitro transmission-blocking studies use a handful of reference laboratory strains, isolated decades ago. While these strains are useful to normalize high throughput screenings, results should be verified on natural parasites that have been selected after years of multiple drug exposures and hence are likely to display differential drug responses compared to reference strains. In addition, it is well known that gametocyte production capacity is lost over time of in vitro culture [2, 4]. Laboratories must therefore rely on precious stocks of cryopreserved isolates with minimal passage since in culture. Adaptation of P. falciparum isolates from patients to in vitro blood-stage culture is routinely performed. Studies describing the use of field isolates for in vitro gametocyte cultures have been published in the early 1980s and have led to the selection of the current reference strains [4–8]. Nowadays using circulating parasites after culture adaptation for transmission-blocking assay is rarely performed. Some studies have shown that they can be used in gametocyte viability assay  or for experimental infections of mosquitoes , however their use for exflagellation-blocking bioassays, reporting in vitro functional maturity of gametocytes has not been reported yet. A requirement for their use in such assay is to be able to produce functional gametocytes in high enough number to be meaningful in a bioassay.
The objective of the work presented here was therefore to determine if culture-adapted field isolates could be used for recently developed in vitro exflagellation-blocking bioassays .
Reference strain and field isolates
The P. falciparum South American chloroquine-resistant strain 7G8 (MRA-926) has been obtained from the MR4. Plasmodium falciparum isolates were collected from mono-infected patients seeking treatment in 2013 in French Guiana (Q206 and Q188) and in 2014 in Cambodia (6831 and 6836).
In vitro culture adaptation
Culture adaptation of isolates was performed using standard protocols [12, 13]. Briefly, after removal of plasma, the red blood cell (RBC) pellet was washed three times in RPMI 1640 supplemented with gentamicin (Gibco-Life Technologies SAS, France) and placed in culture medium (RPMI 1640, 0.5 % AlbuMAX II (Gibco-Life Technologies SAS, France), 2 % decomplemented human plasma) at 4 % haematocrit at 37 °C in 5 or 10 % O2, 5 % CO2, rest of N2 atmosphere. Parasitaemia was checked daily and kept below 2 % by dilutions with fresh RBC and medium. Field isolates were considered adapted to in vitro conditions after three weeks of uninterrupted culture. After culture adaptation, asexual blood-stage sensitivity to anti-malarials was determined using the [3H]-hypoxanthine incorporation assay .
In vitro gametocyte production and maturation
Gametocyte cultures were performed following published protocols [2, 11]. Briefly, asexual cultures with a parasitaemia of ~5 % were used to seed gametocyte cultures at 0.5–1 % parasitaemia and 4 % haematocrit under 5 or 10 % O2, 5 % CO2, rest of N2 atmosphere. Culture medium (RPMI medium with 25 mM HEPES, 50 mg/L hypoxanthine, 2 g/L sodium bicarbonate, 10 % human serum) was replaced daily but without any further addition of RBCs and critically, temperature was maintained at all time at 37 °C.
Gametocytogenesis was evaluated morphologically using Giemsa-stained blood films. Stage V male gametocyte functional maturity was assessed by observation of exflagellation in wet preparation under bright-field microscope. A 30-μL aliquot of gametocyte culture was briefly centrifuged, the cell pellet was resuspended in 15 μL of ookinete medium (RPMI medium with 25 mM HEPES, 50 mg/L hypoxanthine, 2 g/L sodium bicarbonate, 100 μM xanthurenic acid, 10 % human serum) and then introduced into a chamber of a FastRead disposable haemocytometer slide (Immune Systems). Exflagellation centres were observed at 10 × or 20 × magnification.
Exflagellation-blocking assays were performed according to published protocols . Assays were performed using gametocyte cultures providing high enough exflagellation centres for meaningful measures (>30 in five 10 × microscopy fields).
To show the suitability of field isolates for exflagellation-blocking assays, the activity of 1 μM of dihydroartemisinin (DHA), methylene blue (MeBlue) and chloroquine (CQ) was evaluated. Assays were carried out in 1.5-mL tubes containing 170 μL of gametocyte culture medium with 1 μM of drugs dissolved in either DMSO or methanol. Thirty μL of stage V mature gametocyte was dispensed into each assay tube. Tubes were then placed into a 37 °C incubator. After 24 h, exflagellation was induced by temperature drop to 25 °C and replacing culture medium with ookinete medium. The number of exflagellation centres were recorded and compared to controls (DMSO or methanol). Activity was expressed as percentage of exflagellation inhibition compared to controls.
It is important to note that all the experiments using 7G8 and the South American isolates were performed in a different laboratory (Institut Pasteur de la Guyane, Cayenne) than those using the Cambodian isolates (Institut Pasteur du Cambodge, Phnom Penh) showing good reproducibility in the protocols that can be easily implemented in laboratories doing P. falciparum in vitro culture adaptation.
Although the patterns of gametocyte development and time for exflagellation are similar for all isolates and laboratory strains, there are variations in the amount of exflagellation centres from one isolate to another (see Additional file 1) as already observed by others . For example, in French Guiana, the isolate Q206 consistently gave higher number of exflagellation than Q188. Nevertheless, for all the isolates, using gametocyte cultures at maturity, the numbers of exflagellation centres obtained were high enough to allow significant measures in exflagellation-blocking bioassay (from ~35 to >100, see Additional file 1).
Exflagellation-blocking activitya of dihydroartemisinin (DHA), methylene blue (MeBlue) and chloroquine (CQ) against Plasmodium falciparum field isolates and the 7G8 reference strain
99 ± 1.3
90 ± 8.3
78 ± 7.2
84 ± 12.2
79 ± 11.6
99 ± 1.7
99 ± 2.1
−1 ± 10.8
5.7 ± 10.2
7.3 ± 19.2
This work reports that freshly culture-adapted field isolates, from various geographical origins and genetic background can produce functionally mature gametocytes in vitro in high enough numbers to be successfully used in exflagellation-blocking bioassays. Moreover, the assay was sensitive enough to observe inter-strain differences, which might be of significant interest for comprehension of malaria transmission in the field. Indeed, the Q188 isolate is chloroquine-sensitive (IC50 = 34.93 nM ± 1.91) and consistently produced fewer gametocytes in vitro than the Q206 isolate that is chloroquine-resistant (IC50 = 483.36 nM ± 61.25). Of course, given the low number of isolates, no conclusion can be drawn based on this work and further studies are required to evaluate a possible link between chemosensitivity and in vitro gametocytogenesis. In addition, at least one cryopreservation cycle did not impair the capacity of the field isolates in producing mature gametocytes. However, for how long in culture this capacity will be maintained before being lost, as expected remains to be evaluated. Finally, the infectivity to mosquitoes of the mature gametocytes should be assessed to transpose the in vitro results to an in vivo model of transmission. Capacity building for experimental infections of mosquitoes is currently being undergone in Institut Pasteur, both in French Guiana and in Cambodia, and will allow such experiments in a near future.
This work was supported by a European commission REGPOT-CT-2011-285837-STRonGer Grant within the FP7, by an Institut Pasteur ACIP (A10-2013) Grant and by Investissement d’Avenir Grant of the ANR (CEBA: ANR-10-LABX-25-01).
We thank MR4 for providing Plasmodium falciparum 7G8, MRA-926 malaria parasites deposited by Thomas Wellems.
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