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Background   Plasmodium knowlesi  and  Plasmodium vivax  are the predominant  Plasmodium  species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of  P. knowlesi  and  P. vivax , and compare this method to nested PCR and qPCR.    Methods  The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.    Results  The duplex ddPCR assay had higher analytical sensitivity ( P. vivax  = 10 copies/µL and  P. knowlesi  = 0.01 copies/µL) compared to qPCR ( P. vivax  = 100 copies/µL and  P. knowlesi  = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity ( P. vivax  = 80% and  P. knowlesi  = 90%) and clinical specificity ( P. vivax  = 87.84% and  P. knowlesi  = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.    Conclusions  Overall, the ddPCR assay demonstrated acceptable efficiency in detection of  P .  knowlesi  and  P .  vivax , and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay’s clinical sensitivity and specificity.

Droplet digital polymerase chain reaction (ddPCR) for the detection of Plasmodium knowlesi and Plasmodium vivax