Development and evaluation of a pyrosequencing assay for Plasmodium vivax Pvmdr1 genotyping

Reliable detection of Pvmdr1 polymorphisms may support molecular surveillance of genetic variation potentially associated with altered chloroquine susceptibility in Plasmodium vivax. However, conventional genotyping methods may have limited sensitivity for minor alleles in mixed-genotype infections. This study aimed to develop and validate a pyrosequencing assay for combined genotyping of Pvmdr1 single nucleotide polymorphisms. A pyrosequencing assay targeting Pvmdr1 codons 958, 976, and 1076 was developed. Artificial wild‑type and mutant DNA mixtures were used to assess the relationship between expected and observed mutant allele proportions for Y976F and F1076L. Agreement in genotype classification between pyrosequencing and previously generated Sanger sequencing results was assessed using a separate validation set of 49 clinical isolates. The assay was applied to 147 independent P. vivax isolates collected in Kanchanaburi Province, Thailand between 2023 and 2025. The assay showed an approximately linear relationship between expected and observed allele proportions for Y976F (R 2 = 0.9852) and F1076L (R 2 = 0.9829). The lowest nominal mutant proportion detected in all three technical replicates was 30% for Y976F and 10% for F1076L under the tested single-run conditions. Concordance between pyrosequencing and Sanger sequencing was 98.0%, 100.0% and 93.9% at codons 958, 976 and 1076, respectively. Among 147 P. vivax isolates, the M958 mutant genotype classification accounted for 92.5–100.0% of isolates across the three sampling years. The proportion classified as F976 mutant decreased from 21.3% in 2023 to 1.9% in 2025, and F1076L remained common (55.3–62.3%). The three-locus genotype combination (M/Y/L) increased from 34.0% of isolates in 2023 to 79.2% in 2025. This pyrosequencing assay provides a quantitative method for detecting selected Pvmdr1 polymorphisms and mixed-genotype infections. It may support molecular surveillance of P. vivax genetic variation in endemic areas, however, interpretation in relation to chloroquine resistance requires integration with clinical treatment and phenotypic susceptibility data.