Iranian Journal of Medical Microbiology

In vitro Culture of P. falciparum Asexual Stage Parasites

Culture of P. falciparum strain 3D7 was performed at 37°C in human type O⁺ RBCs at 5% haematocrit. Culture medium contained complete medium including RPMI 1640 medium (Sigma-Aldrich), 25 mM HEPES (Sigma-Aldrich), 0.2% D-glucose (Sigma-Aldrich), hypoxanthine 200 μM (Sigma-Aldrich), 0.2% Sodium hydrogen carbonate (Sigma-Aldrich), gentamicin (Invitrogen) 40 mg. L^-1, 0.5% albumex (Invitrogen) and 10% (vol/vol) O⁺ human serum. The parasite was cultured in an atmosphere containing 90% N₂, 5% O₂ and 5% CO₂ (17, 18).
The culture medium was daily gassed and replaced with a new medium (heated to 37°C). Parasite proliferation on each day of the culture period was microscopically examined by taking Giemsa-stained thin blood smear. To synchronize asexual culture, 5% D-sorbitol was used to prepare the parasite in the early trophozoite stage (ring) (19).

Induction of Gametocytogenesis and Gametocyte Culture

 Asexual parasites were cultured to increase the parasitemia to 6-10%. Then the parasitemia was descended to 0.5% (in 6% hematocrit). Cultures were maintained in an atmosphere containing 90% N₂, 5% O₂ and 5% CO₂ without shaking. The cultures were also maintained at 37°C during daily media change. After 72 hours, the hematocrit was reduced to 3% (day 0). Gametocytogenesis was subsequently monitored by daily microscopic examination of the culture medium. On days 6 to 9, asexual forms were removed by treatment with 100 µg. mL^-1 heparin (Sigma-Aldrich). Gametocytes were monitored daily by examining Giemsa-stained thin blood smear until they reached stage V and were prepared for further testing (20).

Quality Control of Functionally Viable Mature Stage V Gametocyte Production and Male Gamete Exflagellation

After production of stage V gametocytes, during daily change of culture medium, the precipitated blood cells were resuspended. 200 μL was taken from the culture medium and quickly transferred to a 1.5 ml tube prewarmed to 37°C. After treating the sample with 50 μM xanthurenic acid in an exflagellation buffer (RPMI 1640 with 25 mM HEPES, 0.2% sodium bicarbonate, pH 8), the culture medium was placed in a microcentrifuge at 2000 g for 30 s at room temperature. Then a thin blood smear was prepared from it and stained with Giemsa and then a light microscope was used to control the quality of exflagellation (21).

Eosin B Test on the P. falciparum Ring Stage

In a 96-well plate, 20 μL of culture containing added asexual parasites was added to all rows except one row belonging to the control group. All samples were repeated in triplicate and placed in a CO₂ incubator for 48 hours.

Eosin B Test on Plasmodium Falciparum Gametocyte

In a 96-well plate, 20 μL of gametocyte-containing culture was added to all rows except one row belonging to the control group. All samples were repeated triplicate and placed in a CO₂ incubator for 42 hours.

Parasitemia and Gametocytemia Evaluation by P. falciparum Lactate Dehydrogenase (PfLDH) Assay

Parasitemia evaluation by lactate dehydrogenase assay is a high-throughput screening assay for antimalarial agents. For this purpose, 100 μL of Malstat reagent (1.57 g Tris HCl, 2 g L-lactic acid, 200 μL Triton X-100 in 85 ml double-distilled water, 66 mg 3-acetylpyridine adenine dinucleotide (APAD), pH 9.1) was added to the 96 –well microplate. Then 20 microliters of infected or uninfected red blood cells were added. The plate was incubated at room temperature and gently shaken for a few minutes to dissolve the red blood cells. During incubation, equal volumes of Nitro Blue Tetrazolium (NBT) and Phenazine Ethosulphate (PES) were mixed away from light and 20 μL of the mixture was added to the wells. The plate was placed away from light. After 30 to 60 minutes, the color change was controlled so that the color tended to dark purple. The plate was read using a BioTek PowerWave XS Microplate Reader at 650 nm. Uninfected red blood cells were used as a reference. Viability percentage was calculated from the following formula (9, 22, 23)
Viability% = 100 x (ODtreated sample – μ_c-) / (μ_c+ - μ_c-μ_c+ = means (μ) of OD control gametocytes (c+ )
μ_c- = means (μ) of OD blank uninfected RBCs (c-)
Statistical analysis: In this study, the tests were repeated 3 times and the results were analyzed by one-way ANOVA at a significance level of p-value <0.05 using GraphPad Prism Version 7.05 software.
 

 Figure 1. Asexual blood stages parasitemia during gametocyte culture.

Figure 2. Different stages of P. falciparum 3D7 during the gametocyte culture period.

Figure 3. Percentage of gametocytemia during gametocyte culture.

Figure 4. The effect of eosin B on the ring of P. falciparum 3D7 parasite. The viability (%) compared to the control group is shown for each test group.
(Means ± SEM (n=5 & P<0.05), One Way-ANOVA test with GraphPad Prism version (7.05)

Figure 5. Comparison of the effect of eosin B (EO) and control drugs chloroquine (CQ) and artemisinin (ART) on P. falciparum 3D7 parasite ring. IC₅₀ values ​​for EO, CQ and ART are shown below each group.  (Means ± SEM (n=5 & P<0.05), One Way-ANOVA test with GraphPad Prism version 7.05)

Figure 6. Effect of eosin B on P. falciparum 3D7 gametocyte.(Means ± SEM (n=5 & P<0.05), One Way-ANOVA test with Graph Pad prism version 7.05)

The Effect of Eosin B on P. falciparum Gametocytes

Figure 6 shows the effect of EO on P. falciparum parasite gametocytes. Compared to the control group, the viability decreased for each test group by increasing the concentration of EO. The IC₅₀ of EO for the parasite gametocyte is 23 nM.
Figure 7 compares the effects of EO and the control drugs CQ and ART on P. falciparum 3D7 gametocytes. The IC₅₀ value of EO is lower than 23 nM compared to standard control drugs. IC₅₀ values for CQ and ART were 41 and 85 nM, respectively. Contrary to the results of the EO test on the blood stages of the parasite, it was observed that EO inhibited the gametocyte growth at a lower dose than standard drugs, indicating a potent inhibitory effect of EO on the P. falciparum 3D7 gametocyte.
 

Figure 7. Comparison of the effect of eosin B (EO) and chloroquine (CQ) and artemisinin (ART) control drugs on P. falciparum 3D7 parasite gametocytes. IC₅₀ values ​​for EO, CQ and ART are shown below each group.(Means ± SEM (n=5 & P<0.05), One Way-ANOVA test with Graph Pad prism version 7.05).
 

Sustained control of malaria is achieved if, in addition to using therapeutic strategies that target the asexual forms that cause the symptoms of malaria, the sexual forms of the parasite, which are the same as gametocytes, are also targeted by drugs that control the parasite transmission stage (24). In the present study, by an improved protocol of using heparin, induction of gametocytogenesis and production of parasite sex cells was performed to test a new compound in the treatment of P. falciparum strain 3D7. It should also be noted that the control of cultured gametocytes should be done carefully so that at least 90% of gametocytes have reached full IV-V growth stage in order to test the drug composition (25). Also, direct comparison of data from studies of drug discovery against gametocytes is difficult due to several factors, including the following: 1- Parasite strain used; 2- Protocol used to induce gametocytogenesis; 3- Combination of culture medium Used; 4- Gametocyte isolation protocols; 5- Developmental stage of gametocytes in the study; 6- Principles of assays used in the experiment; 7- Presence or absence of erythrocytes; 8- Number of gametocytes in each test well; 10- Concentration of tested compounds; 11- Duration of drug administration; 12- How to express the obtained data such as inhibition percentage in a certain concentration or IC₅₀ alone, etc. (14).
EO has shown a significant inhibitory effect on Toxoplasma gondii and the blood stage of P. falciparum. IC₅₀ values for EO in T. gondii and P. falciparum asexual blood stages were 180 μM and 124 nM, respectively (13, 14). Since the IC₅₀ of EO for P. falciparum gametocytes is 23 nM, EO can inhibit gametocytes more severely than asexual blood parasites. Due to the fact that many antimalarial drugs do not have much ability to eliminate the sexual stage of the parasite and even some (such as CQ) induce gametocyte production and increase the number of gametocytes (26), the significant effect of EO on gametocytes and its anti-gammocyte effect in vitro can be considered for the preparation of drugs that block human-to-mosquito transmission.
Methylene blue, which is a dye used in the laboratory, has an inhibitory effect against all stages of P. falciparum (4) and has been proposed as a strong inhibitor of the transition from host to vector (27). Also, its significant effect on gametocytes of this parasite through morphological deformation of gametocytes has been recently reported (28). The IC₅₀ value of methylene blue for P. falciparum gametocyte in vitro is 12.49 nM (24). The IC₅₀ value for EO is close to this combination, indicating the high efficacy of EO against the sexual stage of the parasite.
Compared to the main antimalarial drugs, it can be mentioned that ART, which is an important antimalarial drug, especially in combination therapies, as well as cases of severe and drug-resistant malaria, cannot directly inhibit gametocytes in the patient's body, but it reduces the gametocytemia indirectly by eliminating the blood stages of the parasite (6). The effect of Artesunate, a derivative of ART, on P. falciparum gametocytes has been previously reported in vitro with an IC₅₀ of 102.3 nM (24). Also, primaquine, which is the only drug used clinically to remove gametocytes, has a much higher IC₅₀ in vitro than EO (IC₅₀ 15 μM). Unfortunately, the use of this drug is also limited due to the possibility of hemolytic anemia in people with deficiency in glucose-6-phosphate dehydrogenase (7). Other drugs used clinically against malaria blood stages include quinine and mefloquine, each of which has a higher IC₅₀ than eosin with an IC₅₀ of 50 nM (29). Hydroxychloroquine is another derivative of the important antimalarial drug (CQ) in vitro with an IC₅₀ of 22.78 nM against gametocytes has shown its inhibitory effect (24), which is very close to IC₅₀ EO.  Therefore, the efficacy of EO against the sexual stage of P. falciparum is close to or higher than some of the important known antimalarial drugs. Therefore, due to the possibility of using EO as an oral drug and its strong anti-gametocytic effect compared to some conventional drugs, this combination can be considered as a suitable candidate for use as a drug that blocks the transmission from host to vector.

Given the significant effect of EO on the number of gametocytes and also the comparison of its effect on the blood stage of P. falciparum in vitro, this combination will probably be able to effectively control the transmission from human host to mosquito vector. This effect can be tested by infecting Anopheles mosquitoes, which can be considered in future research using the standard Membrane-Feeding Assay test, which is the gold standard test for blocking transmission to be examined (30). Given the lack of known pharmacological agents that block human-to-vector transmission, identifying the EO compound as an antigamocyte agent could be important for future research.

None.

The authors declared no conflict of interest.