Genetic evidence of cross-border <i>Plasmodium vivax</i> spread in a malaria pre-elimination region of South Asia

Abstract Background Plasmodium vivax is the predominant cause of malaria in South Asia. P. vivax cases have fallen over the past decade, but cross-border transmission remains a major challenge to elimination. Genetic data can generate valuable insights into transmission; however, until now, only low-resolution data have been available from Nepal, Bhutan and Bangladesh. We piloted high-resolution genotyping using a new microhaplotype (multiallelic) assay to monitor P. vivax transmission across borders. Methods Genotyping was conducted using the 93-microhaplotype vivaxGEN panel on P. vivax parasites collected from patients enrolled in clinical trials in Bangladesh, Bhutan, and Nepal between 2013 and 2023. These data were compared with open-access microhaplotype and genomic data derived from Afghanistan, India, and Pakistan between 2014 and 2024. Polyclonality and relatedness (identity by descent (IBD)) were determined within and between countries. Results High-quality genotyping data were generated for Nepal (n = 19), Bhutan (n = 27), and Bangladesh (n = 35); comparative data were sourced from Afghanistan (n = 159), India (n = 24), and Pakistan (n = 213). Overall, 29.6% (47/159) of isolates from Afghanistan and 20.2% (43/213) from Pakistan had polyclonal infections, whereas all parasites from Bhutan, Nepal and Bangladesh were monoclonal, suggesting lower superinfection. Country-wide IBD analyses revealed three genetic clusters partitioning Bangladesh and Bhutan (partial) from the remaining countries. There were two sub-populations in Bhutan, which separated local and cross-border imported cases. Conclusions Our results highlight the use of regional high-resolution genetic data to enhance monitoring of transmission intensity and cross-border importations.