Droplet digital PCR assays for Pvmdr1 and Pvcrt-o gene copy number variation determination in Plasmodium vivax

mdr1 and crt-o amplification in Plasmodium vivax has been associated with antimalarial drug resistance and could thus be a molecular marker. Accurate copy number variation (CNV) quantification could provide valuable insights for monitoring resistance trends.

To develop a duplex digital PCR (ddPCR) assay for the simultaneous absolute pvmdr1 and pvcrt-o quantification using pvtubulin as a single-copy internal reference.

Duplex ddPCR assays were optimized to target pvmdr1/pvtubulin and pvcrt-o/pvtubulin. CNV was calculated as the ratio of the absolute concentration of the target gene to the reference.

ddPCR assays were successfully developed and validated. The quantification limits for assays targeting pvmdr1/pvtubulin and pvcrt-o/pvtubulin, based on pvtubulin concentration, were 22.4 ± 3.5 and 21.5 ± 2.6 copies/µL, respectively. Analysis of field samples from Tak (N = 25) and Kanchanaburi Provinces (N = 97), Thailand, revealed that in 2008, 16% of isolates from Tak carried multiple copies of pvmdr1 and all samples possessed a single copy of pvcrt-o. In contrast, samples collected from the Kanchanaburi Province during 2023-2025 showed no evidence of multiple copies of either gene.

This newly developed ddPCR assay provides a robust and highly reproducible tool for monitoring P. vivax drug resistance markers in endemic regions and a potential approach in surveillance programs.