Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions

Malaria Journal

Methodology

David Poluda¹  Victor Soederstroem¹  Thomas Loescher¹  Nicole Berens-Riha¹  Teferi Eshetu²  Michael Pritsch³  Michael Hoelscher³  Jonathan Shock⁴  Soeren Schubert⁵  Andreas Wieser⁵ 

[1] Division of Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich (LMU), Leopoldstrasse 5, 80802, Munich, Germany;Division of Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich (LMU), Leopoldstrasse 5, 80802, Munich, Germany;Department of Microbiology, Parasitology and Immunology, Jimma University, Jimma, Ethiopia;Division of Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich (LMU), Leopoldstrasse 5, 80802, Munich, Germany;German Centre for Infection Research (DZIF) at LMU, Munich, Germany;Max Planck Institute for Physics, Munich, Germany;Max von Pettenkofer-Institute of Hygiene and Medical Microbiology, Munich, Germany;

关键词: Malaria;    Gametocytes;    Plasmodium falciparum;    Pfs;    Dried blood spots;    Filter paper;    Real-time QT-NASBA;    Storage conditions;    Epidemiology;   

DOI  :  10.1186/1475-2875-11-138

 received in 2011-12-25, accepted in 2012-04-30,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundReal-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.MethodsReal-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 Pfs 25-mRNA due to different storage conditions and time was developed.ResultsPfs 25-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 Pfs 25-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 Pfs 25-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.ConclusionsThe 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.

【 授权许可】

CC BY   
© Pritsch et al.; licensee BioMed Central Ltd. 2012