Dataset Information

Molecular detection and quantification of Plasmodium falciparum-infected human hepatocytes in chimeric immune-deficient mice.

ABSTRACT: BACKGROUND: Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites. METHODS: A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and quantitatively expresses the results as the number of parasites per human hepatocyte. RESULTS: This assay allows for detection of liver stage parasites after challenging humanized mice with infected mosquito bites or after intravenous injection with sporozoites. The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue. CONCLUSIONS: This method allows for the detection and quantification of P. falciparum parasites in chimeric mice repopulated with human hepatocytes. It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

SUBMITTER: Foquet L

PROVIDER: S-EPMC4222492 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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Molecular detection and quantification of Plasmodium falciparum-infected human hepatocytes in chimeric immune-deficient mice.

Foquet Lander L Hermsen Cornelus C CC van Gemert Geert-Jan GJ Libbrecht Louis L Sauerwein Robert R Meuleman Philip P Leroux-Roels Geert G

Malaria journal 20131124

<h4>Background</h4>Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites.<h4>Methods</h4>A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and ...[more]

PMID: 24267791

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Project description:BackgroundThe transmission of malaria to mosquitoes depends on the presence of gametocytes that circulate in the peripheral blood of infected human hosts. Sensitive estimates of the densities of female gametocytes (FG) and male gametocytes (MG) may allow the prediction of infectivity to mosquitoes and thus a molecular estimate of the human infectious reservoir for transmission.MethodsA novel multiplex qRT-PCR assay with intron-spanning primers was developed for the parallel quantification of FG and MG. CCp4 (PF3D7_0903800) transcripts specific for FG and PfMGET (PF3D7_1469900) transcripts specific for MG were quantified in total nucleic acids. The assay was validated on sex-sorted gametocytes from culture material and on samples from clinical trials with gametocytocidal drugs. Synthetic RNA standards were generated for the two targets genes and calibrated against known gametocyte quantities.ResultsThe limit of detection was determined at 0.1 male and 0.1 female gametocyte/µL, which was equal to the limit of quantification (LOQ) for MG, while the LOQ for FG was 1 FG/µL. Results from previously reported clinical trials that used separate gametocyte qRT-PCR assays for FG (targeting Pfs25) and MG (targeting PfMGET) were reproduced with the multiplex assay. High levels of agreement between separate assays and the multiplex approach were observed (R2 = 0.9473, 95% CI 0.9314-0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R2 = 0.8869, 95% CI 0.8541-0.9197, for MG measured by PfMGET in either single or multiplex qRT-PCR). FG and MG transcripts were detected in pure ring stage parasites at 10,000- and 100,000-fold reduced frequency for CCp4 and PfMGET, respectively. The CCp4 and PfMGET transcripts were equally stable under suboptimal storage conditions.ConclusionsGametocyte densities and their sex ratios can be determined in the presented one-step multiplex assay with higher throughput than single assays. The interpretation of low gametocyte densities at asexual parasite densities above 1000 parasites/µL requires caution to avoid false positive gametocyte signals from spurious transcript levels in ring stage parasites.

Dataset Information

Molecular detection and quantification of Plasmodium falciparum-infected human hepatocytes in chimeric immune-deficient mice.

Publications

Molecular detection and quantification of Plasmodium falciparum-infected human hepatocytes in chimeric immune-deficient mice.

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