Sensitive Detection of Foodborne Parasitic Larvae Using a Sieving-qPCR Workflow: Application to Angiostrongylus...

Contaminated food and water represent potential infection sources of Angiostrongylus cantonensis, a parasitic nematode with significant zoonotic potential. Limited environmental surveillance may be due to the absence of simple and reliable screening methods. Different methods of A. cantonensis DNA detection were evaluated on lamb's lettuce (Valerianella locusta). First, three methods for concentrating larvae from water samples spiked with 1 and 10 third-stage larvae (L3) were compared: simple sedimentation and two sieving techniques. Then, two DNA extraction modifications were assessed on spiked lettuce samples. Lastly, the detection limit was evaluated using lettuce samples spiked with 1, 5, 10, 20, and 50 L3. Method efficiency was assessed using a sensitive quantitative polymerase chain reaction (qPCR). The highest DNA yields were obtained using a sieving method with a direct sieve DNA extraction employing glass beads and chelating resin. Using this approach, the detection limit was determined to be a single L3 in 50 g of lamb's lettuce. Detection was successful from both the sieve-retained material and the sedimented flow-through. Sieving techniques provide a promising screening approach for detecting larval stages of parasites, such as A. cantonensis in vegetables. Combined with a sensitive qPCR, detection was consistently achieved at 1 L3 under controlled conditions. Detection of parasite DNA in environmental samples indicates a potential health risk and supports the applicability of this method for food safety monitoring, with possible adaptation to other vegetables.