Development and implementation of droplet digital PCR assays for accurate quantification of Plasmodium vivax parasitemia and G6PD viangchan genotyping

Accurate quantification of Plasmodium vivax parasitemia and identification of glucose-6-phosphate dehydrogenase (G6PD) deficiency, particularly the Viangchan variant, the most prevalent in Southeast Asia, are crucial for effective malaria case management. This study developed, validated, and implemented droplet digital PCR (ddPCR) assays for precise quantification of P. vivax parasitemia and genotyping of the G6PD Viangchan variant. Duplex ddPCR assays targeting the P. vivax tubulin gene and the human RHCE gene were designed to accurately determine parasitemia, with precision and accuracy comprehensively evaluated. In parallel, a duplex ddPCR assay targeting the G6PD Viangchan (871G>A) variant was optimized and assessed for sensitivity and specificity. These assays were applied to clinical samples in Kanchanaburi Province, Thailand. The parasitemia assay demonstrated a limit of detection of 3.2 parasites/µL and a limit of quantification of 400 parasites/µL, at which it exhibited accuracy greater than 90% relative to reference values and coefficients of variation below 20%. For G6PD genotyping, the ddPCR assay clearly distinguished normal, heterozygous, and homozygous/hemizygous individuals, achieving 100% sensitivity and specificity. Parasite densities in clinical samples ranged from 315 to 82,429 parasites/µL. Monitoring parasitemia during chloroquine treatment showed a 99.2-99.9% reduction from day 0 to day 2, with complete clearance by day 3, confirming drug efficacy. G6PD Viangchan genotyping revealed a predominance of the wild-type allele (98.9%), with a single heterozygous female (1.1%) carrying the variant. Collectively, these novel ddPCR assays provide robust tools for accurate parasitemia quantification, treatment monitoring, and rapid, reliable G6PD genetic screening in clinical and epidemiological settings.