2022 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 / Chloroquine |
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| Outline of Annual Research Achievements |
After completing the first step of confirming the chloroquine parasiticidal effect using the laboratory cultured standard strains: chloroquine sensitive (HB3) and resistant parasites: Dd2 (Southeast Asian origin) and 7G8 (South American origin) according to the method of a previous study (Paguio, Mol Biochem Parasitol 2011), the next step was to verify the parasiticidal chloroquine effect using cryopreserved natural parasites recovered from Uganda field site. First by recovering cryopreserved field natural uganda parasites and adapt them to continuous culture in the laboratory. Once successful then perform chloroquine cytocidal drug assay.
Last year, parasite recovery and adaptation into culture failed because the used parasites had been collected in EDTA that hindered parasite growth. We changed the protocol to collect fresh parasite isolates in ACD-A that supports parasite growth.
From Feburary-March 2022 and October-November 2022, two field surveys have been completed in Uganda and 135 fresh parasite isolates (29 in Feb-March 2022 and 106 in Oct-Nov 2022) have been collected. The cryopreserved parasites have been shipped from Uganda to Japan. Parasite recovery and adaptation of these field parasites into continuous culture is ongoing.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Altough we overcame the problem that could have hindered successful parasite recovery and growth, the study is still slightly delayed. This is because until now assessment of the cytocidal effect of chloroquine on the natural field parasites has not yet been done. However, we have completed obtaining fresh natural parasites from Uganda expected to successfully adapt into into culture. There after perform the cytocidal drug assay.
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| Strategy for Future Research Activity |
Parasite recovery and adaptation into culture of the freshly collected natural Uganda parasites is ongoing. After successful culture adaptation of the parasites, the parasiticidal chloroquine effect will be assessed 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 will also be performed. Parasites will be cultured at chloroquine concentrations of 0.025-1.6 μM for 72 hours and IC50 (50% growth inhibitory concentration) evaluated.
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 |
Since my research is delayed, I will use the budget in nex year by following to the plans described in my research proposal.
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