Multiplex qPCR for Detection and Absolute Quantification of Malaria

Abstract

We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R2 values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.

Protection against Plasmodium falciparum malaria by PfSPZ Vaccine

“…AEs and TBSs were assessed at least daily from day 7 to day 18 after CHMI, and then again on days 20, 22, 25, and 28 after initiation of CHMI or until detection of parasitemia as previously described (13). qPCR was done retrospectively (see supplemental materials for specifics) (29,30). Clinical laboratory tests were also monitored.…”

What is the burden of submicroscopic malaria in pregnancy in central India?

“…4,5 A seasoned microscopist is only able to discern parasitemias above a density of approximately 100 parasites per microliter 6,7 compared with PCR where detection can occur at 1 parasite per microliter or less. 8 These low-density parasitemias serve as a reservoir for continued transmission of the disease and may contribute to negative health. [9][10][11] A number of investigators have evaluated the burden of submicroscopic infections during pregnancy in the African subcontinent.…”

Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

“…We isolated P. falciparum from blood samples collected prior to treatment from a total of 108 patients with uncomplicated P. falciparum, obtained during screening for a 2013 dihydroartemisinin (DHA)-piperaquine (PPQ) treatment efficacy study and diagnosed by microscopy and confirmatory Plasmodium species quantitative real-time PCR (12). All patients signed informed consent.…”