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

• Published in: Malaria Journal
• Main Authors: Punitha Mahendran, Jonathan Wee Kent Liew, Amirah Amir, Xiao-Teng Ching, Yee-Ling Lau
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2020
• Subjects: Droplet digital polymerase chain reaction; Plasmodium knowlesi; Plasmodium vivax; Malaria; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/s12936-020-03314-5; https://doaj.org/article/e2cd4a3ef7a043a39ba4b616fdc501f4

Abstract 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.