2021 Fiscal Year Research-status Report
Establishment of novel in vitro assay for evaluation of killing effect of chloroquine against malaria parasite
| Project/Area Number |
20K16238
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| Research Institution | Juntendo University |
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Principal Investigator |
バリカガラ ベテイ 順天堂大学, 大学院医学研究科, 博士研究員 (70805895)
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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Keywords | Plasmodium falciparum / Chloroquine / Cytociday efficacy / Africa |
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| Outline of Annual Research Achievements |
The first objective of the research to confirm the presence of resistance to the parasiticidal effect of chloroquine using laboratory cultured standard parasite strains according to the method of a previous reported study (Paguio, Mol Biochem Parasitol 2011) was confirmed. Using laboratory cultured Plasmodium falciparum parasites; chloroquine sensitive (HB3) and resistant parasites: Dd2 (Southeast Asian origin) and 7G8 (South American origin) we compared the cytocidal chloroquine efficacy (lethal dose 50%, LD50) and conventional cytostatic chloroquine efficacy (inhibitory dose 50%, IC50). The difference of cytostatic value between chloroquine-sensitive HB3 and chloroquine-resistant Dd2 was about 10 times and HB3 versus chloroquine-resistant 7G8 was 5 times. In contrast, 100-fold difference was observed in cytocidal efficacy between HB3 and Dd2 and 10-fold between HB3 versus 7G8.
The next step was to verify the parasiticidal chloroquine effect using cryopreserved natural parasites recovered from Uganda field site. To achieve this objective, the plan was to first recover cryopreserved natural uganda parasites and adapt them into continuous culture in the laboratory. Once parasites are successfully recovered and adapted into culture, then chloroquine cytocidal and cytostatic drug assays were to be performed.
So far, parasite recovery from the cryopreserved state and subsequent adaption into culture has been attempted for 18 Uganda isolates. Unfortunately, the process is not yet successful and is still ongoing.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The progress of this research is slightly delayed because recovery and culture adaptation of the natural field parasites is not yet successful. The problem is thought to arise from the anticoagulant (EDTA) that was used during blood collection of the cryopreserved parasites. EDTA blood anticoagulant seems not to support parasite growth. This could explain the delayed recovery and adaptation into culture of the field parasites. The blood collection protocol has since been changed to using ACD anticoagulant which has no effect on parasite growth.
Fortunately, as of March 2022, we have already collected 29 fresh Uganda parasite isolates into ACD anticoagulant solution. The parasites are awaiting shipment from Uganda to Japan then new drug assays will resume.
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| Strategy for Future Research Activity |
Attempt to recover and adapt into culture of already cryopreserved natural parasites will continue. Subsequent blood collection of malaria parasites will use ACD anticoagulant shown not to have any effect on parasite growth. Following successful culture adaptation of natural parasites, the parasiticidal chloroquine effect will be assessed together with some additional laboratory parasites in two steps.
Step 1: Parasites will be cultured for 6 hours at chloroquine concentrations (0.625-2000 μM) covering the maximum chloroquine concentration in the human body, 10 μM, then the drug will be washed off. Parasites will then be incubated for 48 hours without drug and LD50 (lethal dose concentration) determined from surviving parasites. The conventional in-vitro assay involving exposure of parasites to chloroquine concentrations of 0.025-1.6 μM for 72 hours and determination of IC50 (50% growth inhibitory concentration) will also be performed as the control experiment.
Step 2: The method in step 1 will be improved by developing a new in vitro assay that easily evaluates the parasiticidal chloroquine effect. Specifically, optimize the blood volume used, the chloroquine concentration, drug exposure and culture time, simplify the parasite quantification method, and the objectiveness of the LD50 and IC50 determination methods.
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| Causes of Carryover |
Much of the budget is intended for use to the field sites as well as the laboratory work in Japan. However, money for certain budgeted costs especially travel expenses remains unused because of interruptions arising from the current new corona virus pandemic. Operation of research field sites between Japan and Uganda has been grossly interrupted resulting in some money not being spent.
As the corona virus pandemic wanes, we hope to have the field research sites fully operational.
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Research Products
(5 results)
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[Journal Article] Evidence of Artemisinin-Resistant Malaria in Africa2021
Author(s)
Betty Balikagala, Naoyuki Fukuda, Mie Ikeda, Osbert T Katuro, Shin-Ichiro Tachibana, Masato Yamauchi, Walter Opio, Sakurako Emoto, Denis A Anywar, Eisaku Kimura, Nirianne M Q Palacpac, Emmanuel I Odongo-Aginya, Martin Ogwang, Toshihiro Horii, Toshihiro Mita
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Journal Title
The New England journal of medicine
Volume: 385
Pages: 1163–1171
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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