Figure 1. The detectability of sporozoites by molecular methods and oocysts by immunolabeling.

(A) qPCR performance for P. falciparum sporozoites. Serial dilutions of sporozoites (x-axis) were prepared in phosphate-buffered saline (PBS) and assessed in octuplicate on a single plate to determine qPCR COX-1 limit of detection and quantification. Dots represent sample cycle threshold (left y-axis), and bars coefficient of variation (right y-axis). For each serial dilution, Ct sample positivity is shown as the percentage of total tested. (B) The relationship between oocyst density versus infection prevalence from 457 membrane feeding experiments using cultured gametocytes. Colors represent regular feeds (red) and those selected for performing experiments on the extrinsic incubation period (EIP; green) or sporozoite-expelling experiments (blue). (C) Immunofluorescence staining with 3SP2-Alexa 488 anti-CSP. Empty sheet (left) and intact oocyst (right). (D) Violin plots of oocyst staining – day 8 post infection by mercurochrome (purple) and – day 18 by 3SP2-Alexa 488 anti-CSP immunostaining (yellow) for cultured parasites. Box plots show interquartile range, whiskers show the 95% intervals.

Figure 1—figure supplement 1. qPCR performance on extracted P. falciparum sporozoites (SPZ) targeting the 18S rRNA gene, and DNA extraction/quantification of P. falciparum SPZ from artificial skin.

A serial dilution of SPZ was prepared as described in ‘Materials and methods’ and run in octuplicate to assesses the limit of detection (LOD) and limit of quantification (LOQ) for qPCR targeting 18S. (A) Dots represent sample cycle threshold (left y-axis) and bars the coefficient of variation (right y-axis). For each serial dilution, Ct sample positivity is shown as the percentage of the total number of replicates analyzed. The LOD was set at 100% sample positivity, which was 50 SPZ per sample. The LOQ was set at 100% sample positivity, with a coefficient of variation (COV) <2. For 18S, this was 1000 SPZ per sample (COV 0.71%). (B) Next to test if Integra bovine collagen artificial skin would be suitable as a mosquito feeding membrane to collect expelled SPZ we first tested DNA extraction and quantification from this material. Serial dilutions of SPZ were prepared in phosphate-buffered saline (PBS) and human blood. To compare DNA extraction efficiency, three conditions were tested in duplicate and compared by COX-1 qPCR; SPZ diluted in PBS, SPZ diluted in whole-blood, and SPZ diluted in whole-blood spotted on artificial skin. The amount of SPZ in the sample (x-axis, log 10) plotted against the qPCR Ct value (y-axis) for tested conditions. (C) The amount of SPZ in sample and mean Ct of the three assessed conditions. SPZ serial dilutions in PBS and human blood showed comparable Ct values. SPZ in blood spotted on artificial skin showed slightly lower Ct values, indicating that SPZ can be efficiently extracted from skin without loss of signal. Detection was possible down to approximately five SPZ.