Open Access Highly Accessed Methodology

Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes

Sophie Jones1, Colin J Sutherland1, Cornelus Hermsen2, Theo Arens2, Karina Teelen2, Rachel Hallett1, Patrick Corran1, Marga van der Vegte-Bolmer2, Robert Sauerwein2, Chris J Drakeley1 and Teun Bousema12*

Author Affiliations

1 Department of Immunology & Infection; Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK

2 Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

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Malaria Journal 2012, 11:266  doi:10.1186/1475-2875-11-266

Published: 8 August 2012

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.

Keywords:
Reverse-transcription polymerase chain reaction (RT-PCR); Quantitative nucleic acid sequence-based amplification (QT-NASBA); Sub-microscopic; Gametocyte; Detection; Elimination; Transmission; Ribonucleic acid (RNA); Filter paper