title: Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions.
abstract: BACKGROUND
  Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed.
  
  
  METHODS
  Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed.
  
  
  RESULTS
  Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at -20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per μl.
  
  
  CONCLUSIONS
  The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations.
authors.affiliation: Division of Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich (LMU), Leopoldstrasse 5, 80802 Munich, Germany. pritsch@lrz.uni-muenchen.de
authors.forename: Michael
authors.initials: M
authors.lastname: Pritsch
authors.forename: Andreas
authors.initials: A
authors.lastname: Wieser
authors.forename: Victor
authors.initials: V
authors.lastname: Soederstroem
authors.forename: David
authors.initials: D
authors.lastname: Poluda
authors.forename: Teferi
authors.initials: T
authors.lastname: Eshetu
authors.forename: Michael
authors.initials: M
authors.lastname: Hoelscher
authors.forename: Soeren
authors.initials: S
authors.lastname: Schubert
authors.forename: Jonathan
authors.initials: J
authors.lastname: Shock
authors.forename: Thomas
authors.initials: T
authors.lastname: Loescher
authors.forename: Nicole
authors.initials: N
authors.lastname: Berens-Riha
mesh_terms: D001769:Blood / Q000469:parasitology*; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000469:parasitology*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D021161:Self-Sustained Sequence Replication / Q000379:methods; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors
chemical_list: C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; D012333:RNA, Messenger