When pooling fails: low-intensity Angiostrongylus cantonensis infections in Theba pisana result in false-negativity

Pooling of gastropod samples is frequently used in surveys of the neurotropic nematode Angiostrongylus cantonensis to reduce cost and processing time. Here we highlight a potential limitation of this technique using the snail Theba pisana as an example. Pooled samples tested negative using a high-sensitivity qPCR assay, whereas subsequent individual testing revealed positive cases. This pattern is consistent with a dilution effect, where low parasite DNA concentrations could lead to false-negative results. These findings suggest that pooling may reduce diagnostic sensitivity in regions where infection intensities are low and should be applied with caution.