Abstract
BackgroundThe accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents.MethodsA real-time quantitative PCR (rt-qPCR) method using primers and a hydrolysis probe that targets the 18S rRNA gene was adapted and optimized to estimate parasite load in blood samples. Samples included laboratory prepared blood samples of varying parasite concentrations (6.4 × 105 to 6.4 parasites per 500 μl of packed red blood cells (500pRBC)) and blood samples collected from an experimentally infected human subject collected at 19 time points over 10 days. Sample preparation and extraction, detection chemistry, assay reproducibility, and limit of detection were compared to a previously published SYBR Green rt-qPCR used in a malaria vaccine clinical trial.ResultsBoth the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 101 parasites per 500pRBC. However non-specific amplification in the SYBR Green rt-qPCR assay led to either inaccurate estimation of parasite load at levels below 6.4 × 102 parasites per 500pRBC and to false-positive detection of parasites in negative samples. The rt-qPCR hydrolysis probe assay was specific and provided reliable quantification of parasitaemia down to 6.4 × 101 parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 102 copies per 500pRBC.ConclusionsThese results show that the hydrolysis probe rt-qPCR assay is superior to the SYBR Green rt-qPCR for the quantification of P. falciparum in human blood samples. The hydrolysis probe rt-qPCR is now in use in the Queensland paediatric infectious diseases laboratory (QPID) to monitor parasitaemia in experimentally-infected clinical trial subjects.
Highlights
The accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents
This paper describes the adaptation of this method to allow quantitative monitoring of changes in parasitaemia in human subjects, and compared it to the SYBR Green real-time quantitative PCR (rt-qPCR) method described by Andrews and colleagues [6]
crossing point (Cp) values were reduced by approximately two cycles with the addition of parasite-negative blood nucleic acid extract using the rt-qPCR SYBR Green assay but not in the hydrolysis probe rt-qPCR
Summary
The accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents. A range of diagnostic PCR assays have been developed and these increase the sensitivity of detection of blood-stage malarial parasites by at least a hundredfold compared with traditional microscopy [[3,4], and [5]]. Real-time quantitative PCR (rt-qPCR) assays have been developed for use in malaria vaccine clinical trials, successfully demonstrating quantitative changes in parasitaemia [6]. These methods included a filtration step to remove leukocytes, and human DNA, from the blood during sample preparation, and, have used the intercalating dye SYBR Green for the detection of amplification products
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