A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers

Rockett, Rebecca J., Tozer, Sarah J., Peatey, Chris, Bialasiewicz, Seweryn, Whiley, David M., Nissen, Michael D., Trenholme, Katharine, Mc Carthy, James S. and Sloots, Theo P. (2011) A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers. Malaria Journal, 10 48: 48-1-48-6. doi:10.1186/1475-2875-10-48


Author Rockett, Rebecca J.
Tozer, Sarah J.
Peatey, Chris
Bialasiewicz, Seweryn
Whiley, David M.
Nissen, Michael D.
Trenholme, Katharine
Mc Carthy, James S.
Sloots, Theo P.
Title A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers
Formatted title
A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers
Journal name Malaria Journal   Check publisher's open access policy
ISSN 1475-2875
Publication date 2011-02-28
Year available 2011
Sub-type Article (original research)
DOI 10.1186/1475-2875-10-48
Open Access Status DOI
Volume 10
Issue 48
Start page 48-1
End page 48-6
Total pages 6
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Abstract Background: 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.
Formatted abstract
Background: 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.

Methods:
. A 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 l 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.

Results:
Both 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.

Conclusions: These 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.
Keyword RNA 18S
Protozoal RNA
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 10326
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
School of Medicine Publications
Clinical Medical Virology Centre Publications
 
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Created: Fri, 14 Oct 2011, 02:00:19 EST by Seweryn Bialasiewicz on behalf of Child Health Research Centre