Schistosoma ITS2 Real-Time PCR

DNA extraction and PCR was performed at LUMC as previously described, using a custom automated liquid handling station (Hamilton, Switzerland) [20 (link), 31 (link)]. DNA was extracted from 200 μL of specimen (cervical swab, vaginal swab, CVL) with QIAamp spin columns (QIAGEN Benelux, Venlo, the Netherlands). Detection of the schistosome-specific internal-transcribed-spacer-2 (ITS2) target was performed by real-time PCR as previously described [18 (link), 31 (link)]. This PCR does not differentiate between Schistosoma species. DNA amplification and detection were performed with the CFX96 Real Time PCR Detection System (BioRad, Hercules, CA, USA). The output in threshold cycles (Cts), reflecting the parasite-specific DNA load in the tested sample, was analyzed using BioRad CFX software. Parasite DNA loads were categorized by the following prespecified C_t thresholds: high (C_t < 30), moderate (30 ≤ C_t < 35), low (35 ≤ C_t < 50), and negative (no C_t detected), as previously described [32 (link)].