The online version of this article (doi:10.1186/1475-2875-11-266) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
SJ, TB, CD, CH and RH designed the experiments, RS contributed reagents for the study, MVB cultured the parasites and SJ, TA, KT and CH set up the study. SJ extracted the RNA, performed the RT-PCR and QT-NASBA and CS provided RT-PCR primers and contributed to RT-PCR protocol design. SJ, TB and CD interpreted the data, SJ, TB and PC performed the statistical analysis and SJ and TB wrote the manuscript. CD and CS revised the manuscript. All authors read and approved the final manuscript.
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.
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).
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.
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.
Additional file 1: Table S1. Primer sequences for quantitative nucleic acid sequence based amplification (QT-NASBA) and reverse transcription polymerase chain reaction (RT-PCR). (XLSX 8 KB)12936_2012_2167_MOESM1_ESM.xlsx
Additional file 2: Table S2. Detection by RT-PCR of low-density gametocytes stored on filter papers under a range of incubation conditions. Description of data: Data is represented as percent detection, and number of successes/number of tests, for gametocytes stored on Whatman 903 Protein Saver, the Whatman FTA Classic Card and the Whatman 3MM filter paper for five weeks (top value) and 13 weeks (bottom value). All negative controls were negative (data not shown), NT represents a condition that was not tested. (XLSX 10 KB)12936_2012_2167_MOESM2_ESM.xlsx
Additional file 3: Table S3. Detection by QT-NASBA of low-density gametocytes stored on filter papers under a range of incubation conditions. Description of data: Data is represented as percent detection, and number of successes/number of tests, for gametocytes stored on Whatman 903 Protein Saver, the Whatman FTA Classic Card and the Whatman 3MM filter paper for five weeks (top value) and 13 weeks (bottom value). All negative controls were negative (data not shown), NT represents a condition that was not tested (XLSX 10 KB)12936_2012_2167_MOESM3_ESM.xlsx
Authors’ original file for figure 112936_2012_2167_MOESM4_ESM.pdf
Authors’ original file for figure 212936_2012_2167_MOESM5_ESM.pdf
Authors’ original file for figure 312936_2012_2167_MOESM6_ESM.pdf
Authors’ original file for figure 412936_2012_2167_MOESM7_ESM.pdf
Authors’ original file for figure 512936_2012_2167_MOESM8_ESM.pdf
malERA Consultative Group on Vaccines: A research agenda for malaria eradication: vaccines. PLoS Med. 2011, 8: e1000398- CrossRef
malERA Consultative Group on Vaccines: A research agenda for malaria eradication: drugs. PLoS Med. 2011, 8: e1000402- CrossRef
World Health Organization: Malaria elimination: a field manual for low and moderate endemic countries. 2007, WHO technical report, Geneva
Ouedraogo AL, Bousema T, Schneider P, de Vlas SJ, Ilboudo-Sanogo E, Cuzin-Ouattara N, Nebie I, Roeffen W, Verhave JP, Luty AJ, Sauerwein R: Substantial contribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission. PLoS One. 2009, 4: e8410-10.1371/journal.pone.0008410. PubMedCentralCrossRefPubMed
Schneider P, Bousema JT, Gouagna LC, Otieno S, Vand V, Omar SA, Sauerwein RW: Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection. Am J Trop Med Hyg. 2007, 76: 470-474. PubMed
Carter R, Gwadz RW: Infectiousness and gamete immunization in Malaria. Research in Malaria. Volume 3. Edited by: KJ P. 1980, Academic Press, 263-298.
Shekalaghe S, Drakeley C, Gosling R, Ndaro A, van Meegeren M, Enevold A, Alifrangis M, Mosha F, Sauerwein R, Bousema T: Primaquine clears dubmicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate. PLoSONE. 2007, 2: e1023- CrossRef
Nassir E, Abdel-Muhsin AM, Suliaman S, Kenyon F, Kheir A, Geha H, Ferguson HM, Walliker D, Babiker HA: Impact of genetic complexity on longevity and gametocytogenesis of Plasmodium falciparum during the dry and transmission-free season of eastern Sudan. Int J Parasitol. 2005, 35: 49-55. 10.1016/j.ijpara.2004.10.014. CrossRefPubMed
Ouedraogo AL, Schneider P, de Kruijf M, Nebie I, Verhave JP, Cuzin-Ouattara N, Sauerwein RW: Age-dependent distribution of Plasmodium falciparum gametocytes quantified by Pfs25 real-time QT-NASBA in a cross-sectional study in Burkina Faso. Am J Trop Med Hyg. 2007, 76: 626-630. PubMed
Bousema JT, Schneider P, Gouagna LC, Drakeley CJ, Tostmann A, Houben R, Githure JI, Ord R, Sutherland CJ, Omar SA, Sauerwein RW: Moderate Effect of Artemisinin-Based Combination Therapy on Transmission of Plasmodium falciparum. JInfectDis. 2006, 193: 1151-1159. CrossRef
Schneider P, Bousema T, Omar S, Gouagna L, Sawa P, Schallig H, Sauerwein R: (Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate. Int J Parasitol. 2006, 36: 403-408. 10.1016/j.ijpara.2006.01.002. CrossRefPubMed
Sutherland CJ, Alloueche A, McRobert L, Ord R, Leggat J, Snounou G, Pinder M, Targett GA: Genetic complexity of Plasmodium falciparum gametocytes isolated from the peripheral blood of treated Gambian children. Am J Trop Med Hyg. 2002, 66: 700-705. PubMed
Mlambo G, Vasquez Y, LeBlanc R, Sullivan D, Kumar N: A filter paper method for the detection of Plasmodium falciparum gametocytes by reverse transcription polymerase chain reaction. Am J Trop Med Hyg. 2008, 78: 114-116. PubMed
GE Healthcare UK Limited: Whatman FTATMnucleic acid collection, storage and purification. 2009, Buckinghamshire, UK, technical report 51641
Schneider P, Schoone G, Schallig H, Verhage D, Telgt D, Eling W, Sauerwein R: Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification. Mol Biochem Parasitol. 2004, 137: 35-41. 10.1016/j.molbiopara.2004.03.018. CrossRefPubMed
Pritsch M, Wieser A, Soederstroem V, Poluda D, Eshetu T, Hoelscher M, Schubert S, Shock J, Loescher T, Berens-Riha N: Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions. Malar J. 2012, 11: 138-10.1186/1475-2875-11-138. PubMedCentralCrossRefPubMed
Mens PF, Sawa P, van Amsterdam SM, Versteeg I, Omar SA, Schallig HD, Kager PA: A randomized trial to monitor the efficacy and effectiveness by QT-NASBA of artemether-lumefantrine versus dihydroartemisinin-piperaquine for treatment and transmission control of uncomplicated Plasmodium falciparum malaria in western Kenya. Malar J. 2008, 7: 237-10.1186/1475-2875-7-237. PubMedCentralCrossRefPubMed
Mens P, Spieker N, Omar S, Heijnen M, Schallig H, Kager PA: Is molecular biology the best alternative for diagnosis of malaria to microscopy? A comparison between microscopy, antigen detection and molecular tests in rural Kenya and urban Tanzania. Trop Med Int Health. 2007, 12: 238-244. PubMed
Schneider P, Wolters L, Schoone G, Schallig H, Sillekens P, Hermsen R, Sauerwein R: Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum. J Clin Microbiol. 2005, 43: 402-405. 10.1128/JCM.43.1.402-405.2005. PubMedCentralCrossRefPubMed
Mei JV, Alexander JR, Adam BW, Hannon WH: Use of filter paper for the collection and analysis of human whole blood specimens. J Nutrition. 2001, 131: 1631s-1636s.
Kansagara AG, McMahon HE, Hogan ME: Dry-state, room-temperature storage of DNA and RNA. Nature Meth. 2008, 5: 850-
Dobbs LJ, Madigan MN, Carter AB, Earls L: Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing. Arch Pathol Lab Med. 2002, 126: 56-63. PubMed
Michaud V, Gil P, Kwiatek O, Prome S, Dixon L, Romero L, Le Potier MF, Arias M, Couacy-Hymann E, Roger F, Libeau G, Albina E: Long-term storage at tropical temperature of dried-blood filter papers for detection and genotyping of RNA and DNA viruses by direct PCR. J Virol Methods. 2007, 146: 257-265. 10.1016/j.jviromet.2007.07.006. CrossRefPubMed
De Swart RL, Nur Y, Abdallah A, Kruining H, El Mubarak HS, Ibrahim SA, Van Den Hoogen B, Groen J, Osterhaus AD: Combination of reverse transcriptase PCR analysis and immunoglobulin M detection on filter paper blood samples allows diagnostic and epidemiological studies of measles. J Clin Microbiol. 2001, 39: 270-273. 10.1128/JCM.39.1.270-273.2001. PubMedCentralCrossRefPubMed
- Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes
Colin J Sutherland
Marga van der Vegte-Bolmer
Chris J Drakeley
- BioMed Central
Neu im Fachgebiet Innere Medizin
Meistgelesene Bücher aus der Inneren Medizin
Mail Icon II