Early detection of reduced artemisinin susceptibility in plasmodium falciparum using novel phenotypic indicators

• Main Author: Xiong, Aoli
• Other Authors: Peter Rainer Preiser
• Format: Theses and Dissertations
• Language: English
• Published: 2019
• Subjects: Science::Biological sciences::Microbiology::Drug Resistance
• Online Access: https://hdl.handle.net/10356/106390; http://hdl.handle.net/10220/49591
• Institution: Nanyang Technological University

Artemisinin-based combination therapies (ACT), the first-line anti-malarial treatments recommended by World Health Organization (WHO), have achieved great success globally since their introduction. However, emerging artemisinin resistance in Southeast Asia is posing a real threat as it would have a significant impact on malaria control, treatment and the global elimination efforts. Early and reliable detection of reduced susceptibility to artemisinin is critical for the effective treatment of the disease, as well as surveillance and containment of artemisinin resistant parasites. Currently the most widely used methods for the detection of artemisinin resistance include parasites clearance rate measurements, the ring-stage survival assay (RSA) and DNA based approaches to detect mutations in the K13-propeller gene. All these tests are limited in their usefulness for a rapid and accurate diagnosis due to their intrinsic disadvantages: clearance rate is measured post-treatment; RSA is time-consuming and labor-intensive; test for existing K13 mutations is not only time consuming but may also miss new resistance-conferring mutations. Therefore, the need for an early and reliable phenotypic indicators of artemisinin resistance is compelling. In this thesis, three potential phenotypic indicators were investigated, including the magnetic properties in the magnetic resonance relaxometry (MRR), level of phosphorylated eukaryotic initiation factor 2 α subunit (eIF2α) and the level of artemisinin induced DNA damage. The initial focus of this thesis was to track the magnetic changes in the MRR signal caused by the growth of resistant parasites. An index ΔΔA-ratio was defined and shown to be capable of distinguishing well characterized resistant and sensitive lab strains and one clinical isolate. However, it rendered suboptimal detection accuracy when applied to a wider range of clinical isolates. A new sample preparation approach was later applied and shown to greatly increase the sensitivity of MRR detection. However, this did not improve the detection of artemisinin resistance by MRR. Therefore, alternative approaches focusing on rapid cellular changes triggered by artemisinin were investigated. Phosphorylation of eIF2α and DNA damage were previously shown to be rapidly induced after artemisinin treatment. Their potential as phenotypic indicators of artemisinin resistance were thus investigated. A reproducible reduction in artemisinin-induced phosphorylated eIF2α was observed in multiple resistant parasites compared to their sensitive counterparts, including non-K13-mediated resistant strains, providing a valid basis for further development of rapid diagnostic assays. Alternatively, a lower level of DNA damage was also observed in multiple schizont stage resistant strains compared to the sensitive strains suggesting that DNA damage could serve as another phenotypic marker of resistance. However, this result was complicated by an outlier clinical isolate KH44 that was found to be sensitive to artemisinin but experienced reduced DNA damage. This observation indicated a potential link between the acquisition of artemisinin resistance and DNA damage repair. This link was confirmed by surveying genetic variations in genes involved in DNA repair between resistant and sensitive parasites, providing new insights into broader genetic changes the parasite needs to undergo to acquire artemisinin resistance. In conclusion, this work investigated new phenotypic indicators for early detection of artemisinin resistance. The changes in eIF2α phosphorylation and DNA damage, appearing shortly after in vitro drug treatment, were successfully detected in malaria parasites, and correlated with artemisinin resistance, demonstrating their potentials in future clinical applications.