Last Updated: 11/11/2024
Development of a new diagnostic method for detecting resistant malaria to artemisinin combination therapy
Objectives
The aim of this study is to identify and characterize genes responsible for drug resistance in Plasmodium falciparum, particularly for artemisinin and its combination therapy partner drugs.
The research focuses on two main goals:
- identifying new resistance genes, such as the newly found PfMDR7, using methods like artificial chromosomes and CRISPR/Cas9; and
- developing a reliable resistance detection method based on genetic markers of known and novel resistance genes.
A resistance gene will be identified from the artemisinin-resistant protozoan strain established in this project using an artificial chromosome method. Additionally, the researcher focused on the newly identified resistance gene (PfMDR7) and the known resistance gene (PfMDR1), associated with mefloquine resistance, which is widely used in artemisinin combination therapy. The transcription amount of these genes was used as an index to detect resistance. Furthermore, a resistance detection method will be established by applying the LAMP method, using mutations that confer resistance in known resistance genes (e.g., chloroquine resistance, fancidal resistance) as indicators.
The developed resistance detection method will be tested in Indonesia, taking into account the COVID-19 epidemic situation. In collaboration with experts in drug resistance research in Thailand and Indonesia, the researcher established resistant protozoa derived from malaria patients to study the impact on known resistance genes, employing a unique method involving malaria protozoa artificial chromosomes to search for new resistance genes. Additionally, a diagnostic method for detecting resistant protozoan infections will be developed, using both highly reliable known resistance genes and novel resistance genes as molecular markers.
Blood samples will be collected from Thai falciparum malaria patients to establish a stable culture, with the goal of developing 100 strains. A drug resistance test for artemisinin, mefloquine, piperakin, lumefantrine, and vancidal will be conducted on the stable culture strains to establish patient-derived drug-resistant strains. For isolated resistant strains, the correlation between known drug resistance genes and resistance will be examined by comparing next-generation sequencing results with resistance test outcomes. The detected drug resistance mutations (e.g., point mutations, increased gene copy numbers) will be introduced into drug-sensitive wild-type protozoa using the originally developed CRISPR/Cas9 system to examine resistance conferral.
For isolated protozoans resistant to ACT partner drugs, genomic DNA will be prepared from protozoans lacking a known resistance gene. A gene imparting resistance to ACT partner drugs will be identified using a gene library method with a malaria protozoan artificial chromosome. Additionally, the CRISPR/Cas9 system will be used to confirm the involvement of the identified gene in resistance.
Apr 2018 — Mar 2022
$359,982
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