Why Theba pisana is widely used – and why pooling matters

The terrestrial snail Theba pisana is frequently used in surveys of the zoonotic nematode A. cantonensis due to its high adaptability, abundance, and ease of collection in Mediterranean environments (Jaume-Ramis et al. Reference Jaume-Ramis, Martínez-Ortí, Delgado-Serra, Bargues, Mas-Coma, Foronda and Paredes-Esquivel2023). In general, pooling of gastropod samples is commonly applied to reduce laboratory costs and processing time (Anettová et al. Reference Anettová, Šipková, Baláž, Hambal, Coufal, Kačmaříková, Vanda, Sari and Modrý2025; Furstenau et al. Reference Furstenau, Cocking, Hepp and Fofanov2020). Here, we highlight a potential limitation of this approach, showing how pooling T. pisana individuals during DNA isolation may lead to false-negative results when screening for A. cantonensis in regions where infection intensities are low. This observation underscores the importance of carefully considering pooling strategies in surveillance programmes.

Field sampling and screening approach

During a field survey in Mallorca (Balearic Islands, Spain), 765 T. pisana specimens were collected across the island and screened for A. cantonensis. Due to the large sample size, DNA isolation was performed on pools of five snails (153 pools in total), using foot-tissue subsamples and following the protocol used in Jaume-Ramis et al. (Reference Jaume-Ramis, Martínez-Ortí, Delgado-Serra, Bargues, Mas-Coma, Foronda and Paredes-Esquivel2023). A. cantonensis was detected using the highly sensitive qPCR assay described by Sears et al. (Reference Sears, Qvarnstrom, Dahlstrom, Snook, Kaluna, Baláž, Feckova, Šlapeta, Modry, Jarvi and Nutman2021). Unexpectedly, all pooled samples tested negative.

When pooling fails: evidence from individual retesting

To verify these results, we subsequently tested 30 individual snails from the same pools using individual DNA isolation. While most individuals were negative, a small proportion (three individuals) tested positive, with Ct values typically ≥30, indicating low parasite DNA concentrations. Importantly, these lightly positive individuals originated from pools that had tested negative, indicating that pooling can mask low-intensity infections, consistent with a dilution effect.

Implications for surveillance in low-intensity transmission settings

These findings suggest that T. pisana may act as a relatively poor intermediate host for A. cantonensis in Mediterranean ecosystems, characterised by low infection intensities and low apparent prevalence. Under such conditions, pooling may reduce diagnostic sensitivity, even when using highly sensitive qPCR assays, and may result in misleading conclusions regarding parasite absence. This limitation is not necessarily restricted to T. pisana and is likely to affect surveillance efforts in other gastropod species when infection intensities are similarly low.

We therefore caution against the use of pooled T. pisana samples for A. cantonensis surveillance in Mediterranean regions. While T. pisana remains a suitable target species when analysed individually (Fuentes et al. Reference Fuentes, Gomez-Samblas, Richter, Sáez-Durán, Bueno-Marí, Osuna and Galán-Puchades2024; Martin-Alonso et al. Reference Martin-Alonso, Abreu-Yanes, Feliu, Mas-Coma, Bargues, Valladares and Foronda2015), pooling may represent a suboptimal strategy that risks false-negative outcomes and underestimation of parasite circulation. In this context, we recommend that negative pooled results, particularly in low-intensity settings, should be carefully interpreted and, when possible, validated through individual retesting.

We hope this observation will assist researchers and surveillance programmes in designing sampling strategies that better reflect the ecological context of A. cantonensis transmission.