Improved Protocol for Continuous Culture of Plasmodium falciparum Reference strains

© The Author(s) 2021. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Dominique et al. | J Pure Appl Microbiol | 15(1):123-129 | March 2021 Article 6780| https://doi.org/10.22207/JPAM.15.1.07


iNtROduCtiON
In 2018, the World Health Organization reported 228 million malaria cases with 405,000 deaths. Africa remains the most affected continent with 93% of cases and 94% of deaths 1 . This situation is related to several causes. One of the most important is the resistance of Plasmodium falciparum (P.f.) to the currently used antimalarial drugs such as artemisinin derivatives, amodiaquine, halofantrine, mefloquine, chloroquine and sometimes quinine [2][3][4][5][6] . Indeed, there are 5 parasite species that cause malaria in humans (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi) 7,8 . But only 2 of them (P.f. and P.v.) pose the greatest threat. In 2018, Plasmodium falciparum. accounted for 99.7% of estimated malaria cases in the WHO African Region 50% of cases in the WHO South-East Asia Region, 71% of cases in the Eastern Mediterranean and 65% in the Western Pacific, while P. vivax. is the predominant parasite in the WHO Region of the Americas, representing 75% of malaria cases 9 . Therefore, search for new therapies remains critical. In the process of developing new antimalarial drugs, studies or tests onto the incriminated parasite involve the taking into account of field isolates but also chemo-sensitive or chemo-resistant reference strains for better validation of results. Unfortunately, parasitic biobank of Plasmodium is almost non-existent in Cote d'Ivoire. Indeed, the Biological Resource Center (CeReB), biobank of the Pasteur Institute of Cote d'Ivoire and also for the Sub-Region of ECOWAS countries, was inaugurated only on 25 th April 2019 10 . In addition, acquisition of Plasmodium reference strains from specialized institutions such as BEI Resource, etc. is still very difficult for our local laboratories due to complexity of related administrative procedures. Moreover, even if some research teams have got strains, multiplication attempts using one or the other of the existing protocols came up against many failures such as the low multiplication and the loss of the strains after a maintenance that lasted at most a week. To face all these shortcomings, it was decided in this work to combine several procedures (used to multiply malaria parasites in vitro) into one harmonized protocol to efficiently multiply P. falciparum reference strains.

Laboratory material and reagents
Plasmodium falciparum reference strains used in this study were the NF54 strain sensitive to chloroquine (CQs NF54), and the

Harmonized procedure
The continuous culture of plasmodial strains was based on the procedure described by Trager and Jensen in 1976. Culture medium has been prepared following three protocols. That of the Swiss TPH described by Sergio Wittlin 11 , the "Growing Plasmodium falciparum cultures at high parasitemia" and the "Stockholm sorbitol Thus, the parasites after being removed from the liquid nitrogen were rapidly warmed into a water bath at 37°C and transferred under a laminar flow hood for experiences. Thick film and thin smears have been made to determine parasitemia. Parasites were cultured according to a harmonized protocol (which we have called "CRLP-SwissTPH-Pasteur_001") from the procedures mentioned above, based on several works [14][15][16] . The steps of the work described below, are sum up in the diagram on Fig. 1.

Procedure Preparation of the different media and solutions
500 mL of RPMI (washing) medium will be prepared by measuring 400 mL of sterile distilled water in an Erlenmeyer flask to which 5.217 g of RPMI 1640 powder is added. After dissolving, 3g of HEPES is added, followed by 1g of sodium bicarbonate. The whole is homogenized and the final volume is adjusted to 500 mL with distilled water. The solution obtained is filtered at 0.22µm and 50 mL aliquots are made in conical tubes. To each aliquot 25 µL of gentamycin is added and the whole is stored at -20°C.
Then, 100 mL of albumax-hypoxanthine complex is prepared by measuring 90 mL of RPMI medium (without gentamycin) in an Erlenmeyer flask to which 5g of albumax powder is added. Dissolve and add 20 mg of hypoxanthine. The whole is homogenized and the volume is adjusted to 100 mL with RPMI medium. The solution obtained is filtered a first time at 0.45µm and then a second time at 0.22µm and stored at 2-4°C.
Finally, for this step, an optimized RPMI medium is prepared by measuring 250 mL of RPMI medium (without gentamycin) in an Erlenmeyer flask to which 1.57 mL of L-glutamine, 5 mL of decomplemented human serum, 25 mL of albumax-hypoxanthine complex, 500 mg of D-glucose and 500 mg of sodium bicarbonate are added. The whole is homogenized and filtered at 0.22µm and then 50 mL aliquots are made in conical tubes. Twenty-five (25) µL of gentamicin is added to each aliquot and stored at -20°C.

Thawing the strains followed by culture
The cryotube (containing the plasmodial strain) is taken out of the liquid nitrogen and rapidly heated to 37°C for one to two minutes. The content of the cryotube is then transferred into a 50 mL tube. While homogenizing, a volume of NaCl (12%) equivalent to 0.1 times that of the suspension inside the cryotube is added dropwise and slowly. Incubate at room temperature for five minutes, then slowly add a further volume of NaCl (1.6%) equivalent to 10 times that of the suspension in the cryotube dropwise while homogenizing. Incubate a second time at room temperature for five minutes and then the suspension is centrifuged at 2100 rpm for five minutes. The supernatant is removed and a quantity of washing RPMI equivalent to the volume removed is added. Homogenize again and centrifuge at 1500 rpm for five minutes. The supernatant is again removed and the red cell pellet is adjusted to 200 µL with red cells of the O+ group. An inoculum is then prepared at 2% by adding 10 mL of optimized RPMI medium. This inoculum is homogenized and transferred to a 24 cm 2 culture flask and incubated at 37°C in a CO 2 oven (set at 5% CO 2 + air).

Renewal of the culture medium and preparation of the blood smear slides
Every 48 hours, the flask is carefully removed from the incubator and placed under the laminar flow hood. The supernatant (about 10 mL) Fig. 2. Growing of Plasmodium falciparum NF54 strain is gently removed without touching the blood mat and discarded. The cell mat is slowly homogenized and 15 µL is collected for the blood smear. Then 10 mL of optimized RPMI medium is added to the flask. The solution is gently homogenized and the flask is returned to the CO2 incubator. The blood smears are fixed and stained and the slides are read with a binocular microscope to determine the different parasite densities (DP).

NF54 strain
The CQs NF54 strain responded favorably and more quickly to culture conditions. From day zero (D0) to day four (D4), the parasite density (PD) was multiplied by approximately five; thus, rising from 0.4% to 2.67%. Likewise, from D4 to day eight (D8), this density increased from 2.67% to 11.4%; i.e. approximately 20 times the initial density in eight days of culture (Fig. 2).

K1 strain
Strain CQr K1 reacted more slowly to culture conditions. In fact, a latency time of about one month was observed before perceiving the appearance of schizonts indicating the start of multiplication of the parasite. From this moment reconsidered as D0, up to two days later (D2), the parasite density (DP) increased from 0.1% to 1.58%; ie multiplied by approximately 15. Six days after D2 (ie D8), this density was multiplied by approximately 11, going from 1.58% to 17.07%. Thus, in eight days of culture after awakening of this strain K1, we obtained a parasite density of approximately 170 times the initial one (FIG. 3).

Comment
Overall, we note that this protocol recombination effectively increased the growth of the 2 strains of P. falciparum efficiently. The specific changes made in this protocol boil down to a subsequent modification of the growth medium. Optimization has targeted the association of specific elements related to each protocol; including the additional addition of L-Glutamine, Glucose and Sodium Bicarbonate and then the concomitant presence of decomplemented serum and Albumax-Hypoxanthine complex. Azar and collaborators in 2009, using the Albumax combined with a synchronization alternating Sorbitol and Percoll, were able to obtain 3 to 5 ml of 60% infected blood cells (at an initial parasite density of 10%) in just 1 week of culture17. With the Harmonized protocol, it has been possible to pass from 0.1 to about 17% infected blood cells in a week. In this study, the CQs NF54 strain has been more rapidly reactive to the protocol than the CQr K1 one. Indeed, Mogollon and collaborators in 2016 reported that CQs NF54 strain cultured in standard conditions (RPMI-1640 culture medium supplemented with L-Glutamine and 25mM HEPES (Gibco Life Technologies) + 50 mg/L hypoxanthine (Sigma), supplemented with 10% human serum and 0.225% NaHCO3, at a 5% hematocrit under 4% O2, 3% CO2 and 93% N2 gas-conditions at 75 rpm at 37°C) reacted as well as in the present study18. However, the delay in the reaction of CQr K1 is difficult to explain because it could depend on several factors. One of the elements of the culture medium that could be suspected of being Fig. 3. Growing of Plasmodium falciparum K1 strain a limiting factor in the growth of plasmodia is gentamicin because of its highly hydrophilicity. Nevertheless, Lawrenson et al (2018) showed that the hydrophilicity of some 4-aminoquinolines could therefore optimize activity against CQs NF54 strain, but unfortunately not to overcome chloroquine resistance in CQr K1 strain 19 . As well, the works of Basco et al (2004), Briolant et al (2007) and Duffy et al (2017) highlighted that some culture conditions (such as initial parasitemia, hematocrit, time of incubation, timepoint to add hypoxanthine, use of normal or substitute serum and gas mixture composition) have been singled out as influencing only the in vitro activity assay of drugs but not the ordinary growth of parasites [20][21][22] . But despite all this, CQr K1 has been more productive at the final.

CONClusiON
A mixed protocol from this work that we entitled "CRLP-SwissTPH-Pasteur_001" is available and allows us to efficiently multiply reference P. falciparum strains CQs NF54 and CQr K1. It is planned to spread out the tests to other P. f. strains as well as to wild isolates in order to standardize this process.