Genetic evidence of cross-border plasmodium vivax spread in a malaria pre-elimination region of south Asia

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. Complexity 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). High-complexity infections were observed in 27.04% (43/159) of isolates from Afghanistan, 16.43% (35/213) from Pakistan, and 28.57% (10/35) from Bangladesh. Low prevalence of high-complexity infections was observed in Bhutan (3.70%; 1/27) and Nepal (5.26%; 1/19), suggesting lower superinfection or co-infection. 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.