This report evaluates the stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions. Two methods are widely used to quantify gametocytes in different materials: QT-NASBA and qPCR. There are several advantages of QT-NASBA amplification over qPCR. Primarily, the reverse transcription step to produce complementary DNA (cDNA) is dispensable resulting in faster sample processing and less opportunities to lose RNA or to contaminate samples. As isothermic amplification does not require cycles, amplification time can be reduced [6, 15, 18]. If QT-NASBA could be performed without extraction, this would further speed up sample preparation . The primer attachment sites for qPCR and QT-NASBA can be chosen to be at similar positions within the coding sequence. However, as many genes in Plasmodium do not contain introns, potential cDNA is identical to genomic sequences. Hence, qPCR is totally dependent on complete removal of genomic DNA in contrast to QT-NASBA . In summary, real-time QT-NASBA seems to be more convenient and faster for gametocyte quantification.
Dried blood on filter paper has already been used effectively for gametocyte detection by amplifying specific mRNA . In order to implement this approach in large-scale studies, the estimation of the original gametocyte density should preferably be feasible any time after sample collection - at least up to 3 months afterwards.
No significant differences in detectable mRNA levels for different storage procedures within the first 24 hours were observed. Apparently, temperatures in the range of −20 to 37°C seem to be of little importance for short-term storage as long as the samples have been handled and dried appropriately. Surprisingly, in all DBS subjected to different storage conditions for only 24 hours, significantly more Pfs25-mRNA was detected than in freshly extracted whole blood samples (p = 0.002).
To exclude interference with single-stranded DNA (ssDNA), DNAse- and RNAse- treated samples were amplified. Results indicated no influence of ssDNA. Moreover, it was hypothesised that different makes of filter paper might interfere with the process. So far, the outcome seemed not to be affected by use of four different kinds of filter papers stored for 24 hours at room temperature.
To investigate if fresh whole blood interferes with kit reagents by i.e. inhibiting the extraction or amplification reaction, the whole study was also repeated with gametocyte-spiked whole medium producing similar results. Perhaps the drying process on filter paper might facilitate the lysis of parasites by damaging their walls, resulting in a more efficient lysis than in fresh blood.
By contrast, investigations performed with HIV-1-RNA on DBS demonstrated a significant loss of RNA during the drying process and within the first hours after preparation of filter paper samples compared to fresh whole blood [19, 20]. Perhaps HIV-1-RNA is more accessible in the beginning or degrades faster during the drying process.
In conclusion, the correction factor for signal increase was found to be stable in the specific study settings and was not influenced by the tested storage procedures. It is however recommended to determine the individual correction factor depending on the workflow used for each study.
After 28 days, significant differences between the storage conditions could be observed. As expected, the freezer (−20°C) prevented most efficiently RNA decay (average recovery rate 98.7%) and 37°C performed worst (average recovery rate 66.7%) (see also Figure 1). Similar results could be obtained after 92 days of storage. At day 28, storage in the fridge showed a slight, but insignificant, trend towards higher decay rates. This trend was only found once in the experiment selected for presentation and not in the other repetitions. It can be assumed that this trend was due to statistical variation. The experiment was neverthless selected as it was the most extensive performed with storage times of up to 92 days (transport simulation, room temperature and freezer).
Exponential decay rates for each storage condition were calculated, allowing estimates of signal loss experienced for each individual sample as long as storage conditions and duration were known. Naturally, all calculations rely on standardised procedures and might vary depending on the individual setting.
The theoretical limit of detection (LOD) was calculated by linear regression with over 42 duplicates of 24 h-old DBS. A density of less than 10 gametocytes per ml could be detected (0.0096/μl, 95% CI 0.0025 to 0.038). Nevertheless, due to methodical non-linearity of the QT-NASBA reaction for target concentrations with absolute extremes, the linearity of regression is only an approximation. LODs close to one gametocyte per DBS are subject to huge dilution effects and stochastic noise. In addition, the LOD will decrease with prolonged storage due to target decay. However, an individual reasonable LOD can be estimated with the data presented above.
QT-NASBA amplification of DBS was performed from Ethiopian patients, who were tested negative for gametocytes by microscopy in 2008 and 2009. Interestingly, 22 (69%) of 32 cases were detected positive for gametocytes nevertheless with QT-NASBA (Additional file 4). These results were astonishing, as the samples had been stored for 2–12 months at room temperature and subsequently 18–30 months at −20°C. Significant decay had to be expected. However, successful RNA extraction has also been described after long-term storage if the initial load was high enough .
Although it is unlikely that a mosquito blood meal of 2–3 μl at gametocyte densities below 0.20-0.30 gametocytes per μl contains at least one male and one female gametocyte required for fertilisation , Schneider and colleagues could prove that some patients tested negative for Pfs25-mRNA with QT-NASBA could still infect mosquitoes . It is yet to be investigated to what extent these very low-level gametocyte densities contribute to the infectious reservoir and at what frequency they result in mosquito infection. Sub-microscopic gametocyte carriage over longer time scales is known to play an important role in sustaining transmission .