DNA was extracted from 100 μL blood using the Genomic DNA Extraction kit (Macherey-Nagel, Düren, Germany) and eluted in an equivalent volume of elution buffer. DNA was screened for P. falciparum using ultrasensitive qPCR that amplifies a conserved region of the var gene acidic terminal sequence (varATS) according to a previously published protocol [41 (link)]. The varATS gene assay amplifies ~ 20 copies/genome [41 (link)]. The qPCR results were converted to varATS copies/μL using external standard curve of ten-fold serial dilutions (5-steps) of 3D7 P. falciparum parasites quantified by droplet digital PCR (ddPCR) [42 (link)]. The ddPCR thermocycling conditions, sequences and concentration of primers and probe are given in additional file 1 . Asexual parasite densities were calculated by dividing varATS copy numbers by 20, reflecting the approximate number of copies per genome.
For all the gametocytes assays, RNA was extracted using the pathogen Nucleic Acid Extraction kit (Macherey-Nagel, Düren, Germany) and eluted in 50 μL elution buffer, i.e., RNA was concentrated two-fold during extraction. RNA samples were DNase treated (Macherey-Nagel, Düren, Germany) to remove genomic DNA that could result in a false positive pfs25 signal [43 (link)]. A subset of RNA samples was tested by varATS qPCR, and all tested negative.
For all the gametocytes assays, RNA was extracted using the pathogen Nucleic Acid Extraction kit (Macherey-Nagel, Düren, Germany) and eluted in 50 μL elution buffer, i.e., RNA was concentrated two-fold during extraction. RNA samples were DNase treated (Macherey-Nagel, Düren, Germany) to remove genomic DNA that could result in a false positive pfs25 signal [43 (link)]. A subset of RNA samples was tested by varATS qPCR, and all tested negative.
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