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

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 tes...

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Bibliographic Details
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
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Summary: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.

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