Interrupting the transmission of malaria has become a priority as we move towards elimination1. To design and test transmission blocking strategies we need a good understanding of malaria transmission dynamics. Transmission can occur at extremely low, submicroscopic gametocyte densities2; therefore sensitive methods are required for detection and quantification of both male and female gametocytes. Traditional microscopy is limited by sensitivity and specificity3. Although RT-qPCR offers increased sensitivity, reporting of quantitative data can vary between laboratories due to a range of reasons, including the standards used for quantification. Droplet digital PCR (ddPCR) offers precise quantification without the need for external reference standards, and is therefore potentially useful for improving inter-laboratory comparison4. In this pilot study, we explored the use of ddPCR for quantifying gametocytes.
Two gametocyte markers (female: pfs25 and male: pfMGET) were assessed. Purified gametocytes, synRNA standards and clinical trial samples were analysed by both ddPCR and RT-qPCR, and assay performance characteristics (quantitative correlation, sensitivity and reproducibility) compared. Both assays were also used to determine the number of pfs25 or pfMGET transcripts per gametocyte to allow quantification of gametocyte numbers.
ddPCR quantitative results showed excellent agreement with RT-qPCR for clinical samples (r>0.98, p<0.0001, mean difference: pfs25 -0.1853 log10 gametocytes/ml and pfMGET -0.1677 log10 gametocytes/ml against RT-qPCR). ddPCR showed similar sensitivity (0.4-1.2 copies/ul) and reproducibility (Bartlett’s p>0.05) to RT-qPCR when testing replicates of purified gametocyte and synRNA standards. Moreover, numbers of pfs25 or pfMGET transcripts detected per gametocyte using ddPCR and RT-qPCR were comparable and estimated to be 265 or 280 pfs25 copies/female gametocyte and 20 or 13 pfMGET copies/male gametocyte.
These preliminary data show that ddPCR offers similar assay performance characteristics to our established RT-qPCR methods. It can be used where absolute quantification is needed without reference standards and has the potential for improving inter-laboratory comparison.