Abstract
The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.
Highlights
Malaria is a vector-borne disease, caused by the apicomplexan parasite Plasmodium spp. and transmitted by Anopheles mosquitoes
The low percentage of host hepatocytes that are infected after inoculation with Plasmodium spp. sporozoites and the limited number of available tools pose major obstacles for the evaluation of liver-stage development and the quantitative analysis of pre-erythrocytic anti-parasite effects induced by experimental drug treatments or vaccines in vivo [3]
The parasite liver burden in samples derived from P. yoelii infected or uninfected mice was quantified by calculating the ratio of ‘plasmid equivalents’ of P. yoelii 18S rRNA (Py18S) rRNA or PyCytB mitochondrial DNA (mtDNA) to 106 units of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Summary
Malaria is a vector-borne disease, caused by the apicomplexan parasite Plasmodium spp. and transmitted by Anopheles mosquitoes. The low percentage of host hepatocytes that are infected after inoculation with Plasmodium spp. sporozoites and the limited number of available tools pose major obstacles for the evaluation of liver-stage development and the quantitative analysis of pre-erythrocytic anti-parasite effects induced by experimental drug treatments or vaccines in vivo [3]. The complete absence of blood-stage parasitaemia indicates sterile protection Such assessment fails to evaluate any effects directed only at the liver stage or discriminate between the liver and blood stage for interventions potentially directed at either or both parasite stages. We and others have previously described qRT-PCR methods which can detect liver stage parasite burden following challenge with relatively high doses of sporozoites or bites of infected mosquitoes.
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