Universal Blood Parasite Detection

DNA from 200 μL of parasite-free or parasite-infected whole blood was extracted using a QIAamp DNA Blood Mini QIACube Kit (Qiagen) and a Qiagen QIACube for automated sample preparation. Samples and negative extraction controls were processed according to the kit protocol and eluted into 50 μL of Qiagen Buffer EB. Following extraction, 8.5 μL of DNA extract was digested for 2 h at 37 °C with 10 units of PstI (0.50 μL) and 1 μL of 10X CutSmart Buffer (digest D1). Digested DNA (2 μL of that digest) was subsequently amplified (PCR1) in a volume of 25 μL per reaction. For PCR1 cycling, samples were denatured at 98.0 °C for 30 s followed by 15 cycles of 98.0 °C for 10 s, primer annealing at 67.0 °C for 30 s and extension at 72.0 °C for 2 min, and a final extension at 72.0 °C for 2 min. Thereafter, the entire 25 μL product of PCR1 was again digested for 1 h at 37 °C by directly adding 10 units of BamHI-HF (0.5 μL), 10 units of BsoBI (1 μL), and 2.5 μL of 10X CutSmart Buffer (digest D2) to the original PCR tube. Two microliters of that digested product were then transferred into PCR2 (20 μL per reaction). All restriction enzymes were purchased from NEB (Ipswich, MA, USA). For PCR2, samples were denatured at 98.0 °C for 30 s followed by 30 cycles of 98.0 °C for 10 s, primer annealing at 67.0 °C for 30 s and extension at 72.0 °C for 45 s, and a final extension at 72.0 °C for 2 min. All PCR reactions (both PCR1 and PCR2) contained Q5 Buffer, dNTPs, forward and reverse primers (1.25 μL of 10 μM stock), Q5 High GC Enhancer, and Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA), in the concentrations specified in the manufacturer’s instructions. Only the reaction volumes varied between PCR1 (25 μL) and PCR2 (20 μL). Each PCR run was accompanied by at least three negative controls—DNA extracts of parasite-negative blood. To establish the conditions described here, assay parameters were optimized systematically using DNA from cultures of Leishmania infantum spiked into human blood and P. knowlesi-containing Macaca mulatta blood; optimal conditions were considered to be those that most reduced the number of vertebrate-derived reads in the final sequence dataset while maximizing the number of parasite-derived reads. An overview of these optimization experiments is provided in Supplementary File S1. Specimens were processed using the method described here in addition to comparing different restriction digestion procedures: specimens prepared using only Digest 1, only Digest 2, or both digestion steps. Each of these three reaction conditions was applied to the same specimens in triplicate and sequenced on three separate MiSeq sequencing runs. These results were compared to a single replicate prepared for each specimen analyzed using the method described by Flaherty et al. [18]. The significance of differences between all conditions were assessed using a 2-way ANOVA with Tukey’s multiple comparisons posttest.