title: New molecular settings to support in vivo anti-malarial assays.
abstract: BACKGROUND
  Quantitative 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.
  
  
  METHODS
  FTA 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. noemi.r.bahamontes-rosa@gsk.com.
authors.forename: Noemí
authors.initials: N
authors.lastname: Bahamontes-Rosa
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. ane_rodriguez@hotmail.com.
authors.forename: Ane Rodriguez
authors.initials: AR
authors.lastname: Alejandre
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. vanesa.j.gomez@gsk.com.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Gomez
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. sara.m.viera@gsk.com.
authors.forename: Sara
authors.initials: S
authors.lastname: Viera
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. maria.g.gomez@gsk.com.
authors.forename: María G
authors.initials: MG
authors.lastname: Gomez-Lorenzo
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. laura.x.sanz@gsk.com.
authors.forename: Laura María
authors.initials: LM
authors.lastname: Sanz-Alonso
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain. alfonso.x.mendoza@gsk.com.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Mendoza-Losana
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000097:blood / Q000302:isolation & purification; D057230:Limit of Detection; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology*; D051379:Mice; D010274:Parasitology; D010961:Plasmodium / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D016054:DNA, Protozoan