The online version of this article (doi:10.1186/1475-2875-11-138) contains supplementary material, which is available to authorized users.
Michael Pritsch, Andreas Wieser contributed equally to this work.
The authors declare that they have no competing interests.
VS, MP, NBR, MH, SS and AW designed the experiments. MP, VS, DP and NBR performed real-time QT-NASBA and cultured parasites. MP, TE and NBR performed microscopy. TE, NBR and LT provided clinical samples. JS, MP, VS, AW and NBR performed data analysis. MP, VS, DP, NBR, JS and AW wrote the manuscript. All authors read and approved the final manuscript.
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.
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 Pfs 25-mRNA due to different storage conditions and time was developed.
Pfs 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.
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.
Additional file 1: Calculated retrieval rates of Pfs 25-mRNA in a hypothetical storage scenario. The measured decay rates for individual storage procedures were used to derive a formula, which simulates retrieval rates of Pfs 25-mRNA after certain time periods. The figure presents the storage simulation using a backpack (one week), a fridge (one month) and finally a freezer (two months). Ultimately an average recovery rate of 48.0% would be achieved. (PDF 38 KB)12936_2011_2171_MOESM1_ESM.pdf
Additional file 2: Comparison of DNAse and RNAse treated samples from a 24 h old extracted DBS stored at room temperature. It is known that ssDNA can be amplified in an RNA-based NASBA setting [ 14, 15], therefore additional experiments were performed. Standard filter paper (Whatman Chromatography 3MM) spotted with a 50 μl blood sample of 0.28 gametocytes/μl (average of 14 gametocytes in the whole spot) was extracted. The eluate of nucleic acid extraction was then separated into three portions. One was treated with DNAse (DNAse I,1U/μl, Fermentas) and the other with RNAse (RNAse A, 10 mg/ml, Fermentas) respectively. The third sample was stored next to the treated tubes in the shaker and heat block and served as a control. The enzymatic treatment enables a direct comparison of amplification from DNA and RNA free samples. Incubations at 37°C for 15 minutes followed by 75°C for 20 minutes to inactivate the enzymes was required. As expected, the heat treatment reduced the signal amplitude slightly compared to other control samples incubated on ice, probably due to nucleic acid decay during incubation at the higher temperatures. However, it could be demonstrated, that the time to positivity measured by QT-NASBA did not differ between the DNAse treated samples and the heat treated control and no signal could be acquired from the RNAse treated samples. This data excludes the presence of plasmodium ssDNA or dsDNA as cause for the signal increase experienced after 24 h from filter paper. The same experiment was also performed with extracted whole blood dilutions and confirmed the results. (PDF 61 KB)
Additional file 3: Comparison of different types of filter papers in the ability for Pfs25-mRNA retrieval. In order to evaluate how different additives and qualities of filter papers actually affect the stability of RNA, standard Whatman Chromatography paper without impregnation (3MM Chr, Catalog number 3030 917) was used as a reference compared to the FTA classic cards (catalog number WB120205) [Tsumori et al 2011], FTA Micro Card (catalog number WB120210) and the 903 Protein Saver Card (catalog number 10534612) [Shekalaghe et al 2011]. Filter papers were spotted with 50 μl fresh whole blood containing 2.25 gametocytes/μl, dried and kept in sealed plastic bags at room temperature. RNA extraction and immediate NASBA amplification after 24 h of storage did not show any differences between the different makes. (PDF 62 KB)12936_2011_2171_MOESM3_ESM.pdf
Additional file 4: Clinical DBS samples(PDF 74 KB)12936_2011_2171_MOESM4_ESM.pdf
Authors’ original file for figure 112936_2011_2171_MOESM5_ESM.pdf
Authors’ original file for figure 212936_2011_2171_MOESM6_ESM.pdf
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- Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions
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