Open Access Highly Accessed Methodology

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

Michael Pritsch12*, Andreas Wieser3, Victor Soederstroem1, David Poluda1, Teferi Eshetu14, Michael Hoelscher12, Soeren Schubert3, Jonathan Shock5, Thomas Loescher1 and Nicole Berens-Riha1

Author Affiliations

1 Division of Infectious Diseases and Tropical Medicine, Medical Center of the University of Munich (LMU), Leopoldstrasse 5, 80802, Munich, Germany

2 German Centre for Infection Research (DZIF) at LMU, Munich, Germany

3 Max von Pettenkofer-Institute of Hygiene and Medical Microbiology, Munich, Germany

4 Department of Microbiology, Parasitology and Immunology, Jimma University, Jimma, Ethiopia

5 Max Planck Institute for Physics, Munich, Germany

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Malaria Journal 2012, 11:138  doi:10.1186/1475-2875-11-138

Published: 30 April 2012

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.

Keywords:
Malaria; Gametocytes; Plasmodium falciparum; Pfs25-mRNA; Dried blood spots; Filter paper; Real-time QT-NASBA; Storage conditions; Epidemiology