Advancing artemisinin resistance monitoring using a high sensitivity ddPCR assay for Pfkelch13 mutation detection in Asia.

The spread of Pfkelch13 mutations in Southeast Asia threatens the effectiveness of artemisinin-based combination therapies (ACTs) for malaria. Previous studies revealed a high prevalence of key mutations, including C580Y, P574L, and R561H, emphasizing the need for the surveillance to combat drug resistance. This study, we developed a droplet digital PCR (ddPCR) assay for the rapid screening of common mutations including P441L, Y493H, P527H, G538V, R539T, I543T, R561H, P574L, C580Y, and A675V. The assay was designed to detect minor populations of mutant strain within multiple infection, offering high sensitivity and specificity using artificial mixtures of mutant and wild-type alleles. Field samples collected in Thailand during 2015-2020 and in 2023 (N = 130) were also analyzed to validate the assay in a real-world setting. The ddPCR assay demonstrated exceptional performance, with 100% sensitivity and 90% specificity. The R539T, R561H, and C580Y mutations were detected in clinical samples collected from several study sites in Thailand. Notably, the R561H mutation was detected in 100% of the P. falciparum isolates from Mae Hong Son, Thailand in 2023, underscoring the assay's utility in identifying critical mutations associated with drug resistance. Moreover, ddPCR can detect multiple parasite populations in clinical samples and can be used to analyze the ratios of wild-type and mutant alleles. These results validate the assay's ability to serve as a powerful tool for the early detection of minor allele frequencies, facilitating the timely implementation of interventions to curb the spread of ACT resistance. The ddPCR assays developed in this study provide a sensitive and specific method for detecting Pfkelch13 mutations, allowing the identification of minor parasite populations with artemisinin resistance. These assays enhance our ability to monitor and respond to malaria drug resistance, offering a crucial tool for early detection and contributing to global malaria elimination efforts.