title: Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes.
abstract: BACKGROUND
  Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored.
  
  
  METHODS
  Three gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR).
  
  
  RESULTS
  Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p<0.0001). When papers were stored at higher temperatures, a loss in sensitivity was experienced for the FTA Classic Card but not the 903 Protein Saver Card or Whatman 3MM filter paper. The sensitivity of gametocyte detection was decreased when papers were stored at high humidity.
  
  
  CONCLUSIONS
  This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.
authors.affiliation: Department of Immunology & Infection; Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.
authors.forename: Sophie
authors.initials: S
authors.lastname: Jones
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Sutherland
authors.forename: Cornelus
authors.initials: C
authors.lastname: Hermsen
authors.forename: Theo
authors.initials: T
authors.lastname: Arens
authors.forename: Karina
authors.initials: K
authors.lastname: Teelen
authors.forename: Rachel
authors.initials: R
authors.lastname: Hallett
authors.forename: Patrick
authors.initials: P
authors.lastname: Corran
authors.forename: Marga
authors.initials: M
authors.lastname: van der Vegte-Bolmer
authors.forename: Robert
authors.initials: R
authors.lastname: Sauerwein
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Drakeley
authors.forename: Teun
authors.initials: T
authors.lastname: Bousema
mesh_terms: D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000469:parasitology*; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods; D010274:Parasitology / Q000191:economics / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification*; D013048:Specimen Handling / Q000191:economics / Q000379:methods*
chemical_list: D012333:RNA, Messenger