New molecular settings to support in vivo anti-malarial assays

Abstract

BackgroundQuantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods.MethodsFTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes.ResultsThe optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed.ConclusionsThis is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process.

Plasmodium exoerythrocytic parasites redirect trafficking of human proteins to the parasitophorous vacuole

“…Quantification of Pb -Luc infection was as previously described 72 . Briefly, 90,000 HC-04 HsNR4A3 WT and CRISPR/ Cas 9 edited cells were plated 24h before infection in 24-well plates.…”

“…These clones were then used for further studies. berghei sporozoites, fixed at different times (3,6,12,18,24,48,72, and 96 hours). Cells were labeled using a α-HsNR4A3 (#TA804893) and visualized with a goat α-mouse secondary antibody (Rhodamine -red); CellMask deep red was used for plasma membranes (Cell Membranemagenta).…”

“…We thank the members of the Winzeler laboratory for advice and critical reading of the manuscript. In addition, we thank Medicines for Malaria Venture and Fogarty International Center berghei sporozoites, fixed at different times (3,6,12,18,24,48,72, and 96 hours). Cells were labeled using a α-HsNR4A3 (#TA804893) and visualized with a goat α-mouse secondary antibody (Rhodamine -red); CellMask deep red was used for plasma membranes (Cell Membranemagenta).…”

Beyond the microcirculation: sequestration of infected red blood cells and reduced flow in large draining veins in experimental cerebral malaria

“…with and without pyrimethamine-treated mice employing the respective Taqman gene expression assay (Applied Biosystems, Germany). To determine the parasite load in the different regions of the brain, q-PCR was performed for P. berghei ANKA cytochrome B (PBANKA_MIT01900) 72 and HPRT (Eurofins Genomics, Germany) using Light cycler 480 SyBr Green I master mix (Roche, Germany). Amplification was performed with a Light cycler 480 (Roche, Germany).…”

“…For registration of the TOF template to standard space, the T2-weighted (T2w) image was rigidly registered to the Allen brain template, and segmented into the three tissue compartments (gray matter, white matter, and cerebrospinal fluid) using SPM’s ( https://www.fil.ion.ucl.ac.uk/spm/ ) unified segmentation approach 78 . The tissue segments where further used for affine and subsequent nonlinear registration to the template tissue probability maps 79 using elastix 72 to obtain the T2w image in standard space. Using the concatenated transformation parameters from rigid, affine, and nonlinear registrations, the TOF template was finally transformed to standard space.…”