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

ABSTRACT: 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 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 × 10(5) 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.Both the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 10(1) 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 × 10(2) 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 × 10(1) parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 10(2) copies per 500pRBC.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.

SUBMITTER: Rockett RJ

PROVIDER: S-EPMC3055851 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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

Rockett Rebecca J RJ Tozer Sarah J SJ Peatey Chris C Bialasiewicz Seweryn S Whiley David M DM Nissen Michael D MD Trenholme Katharine K Mc Carthy James S JS Sloots Theo P TP

Malaria journal 20110228

<h4>Background</h4>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.<h4>Methods</h4>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 concentra ...[more]

PMID: 21352599

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Project description:BackgroundAvian malaria parasites (genus Plasmodium) are cosmopolitan and some species cause severe pathologies or even mortality in birds, yet their virulence remains fragmentally investigated. Understanding mechanisms and patterns of virulence during avian Plasmodium infections is crucial as these pathogens can severely affect bird populations in the wild and cause mortality in captive individuals. The goal of this study was to investigate the pathologies caused by the recently discovered malaria parasite Plasmodium homocircumflexum (lineage pCOLL4) in four species of European passeriform birds.MethodsOne cryopreserved P. homocircumflexum strain was multiplied and used for experimental infections. House sparrows (Passer domesticus), common chaffinches (Fringilla coelebs), common crossbills (Loxia curvirostra) and common starlings (Sturnus vulgaris) were exposed by subinoculation of infected blood. Experimental and control groups (8 individuals in each) were observed for over 1 month. Parasitaemia, haematocrit value and body mass were monitored. At the end of the experiment, samples of internal organs were collected and examined using histological and chromogenic in situ hybridization methods.ResultsAll exposed birds were susceptible, with similar average prepatent period and maximum parasitaemia, yet virulence was different in different bird species. Mortality due to malaria was reported in chaffinches, house sparrows and crossbills (7, 5 and 3 individuals died respectively), but not in starlings. Exoerythrocytic meronts (phanerozoites) were observed in the brain of all dead experimental birds. Blockage of blood vessels in the brain led to cerebral ischaemia, invariably causing brain damage, which is likely the main reason of mortality. Phanerozoites were observed in parenchymal organs, heart and muscles of all infected individuals, except starlings.ConclusionThis study shows that P. homocircumflexum is generalist and the same lineage caused similar parasitaemia-related pathologies in different host species. Additionally, the mode of exo-erythrocytic development is different in different birds, resulting in different mortality rates. This should be taken into consideration in studies addressing pathology during avian malaria infections.

Dataset Information

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

Publications

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

Similar Datasets

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