Development of a Molecular Approach for Surveillance of Angiostrongylus cantonensis in Preserved Host Gastropod Collections

Abstract

Angiostrongylus cantonensis (rat lungworm) is a neurotropic zoonotic nematode and the leading cause of eosinophilic meningitis globally. Since its first description in China in 1935, the parasite has expanded well beyond its native range into Southeast Asia (eg: Thailand, Cambodia, Indonesia, and Philippines), the Pacific islands (eg: Fiji, Micronesia, and Polynesia), South America (eg: Brazil and Ecuador), and is now established in Hawaii and parts of the continental United States (eg: Oklahoma, Texas, and California), where it has emerged as a growing One Health concern involving human, animal, and environmental health. In Hawaiʻi, several isolated, as well as consecutive, years have shown elevated reported case counts, indicating a need to investigate whether changes in ecological transmission, particularly parasite prevalence in gastropod hosts, may explain current disease dynamics.This central role of gastropods in transmission raises an important methodological question: whether A. cantonensis DNA shed from infected hosts can be detected from preservative ethanol and associated sediment, thereby supporting minimally destructive environmental surveillance by only sacrificing the gastropods but keeping their tissue intact. Ethanol-preserved gastropod samples are particularly valuable for this purpose because they are commonly generated during field collection and storage, and the preservative fluid may accumulate parasite-derived DNA together with host-associated material such as mucus, fecal residue, food debris, and other environmental particulates carried by the gastropods, forming into sediment. Our study evaluates a molecular detection of eDNA for A. cantonensis in gastropod-associated preservative ethanol and sediment. We postulate that the infected gastropods carrying parasite within them may shed A. cantonensis DNA into the surrounding preservative ethanol, and that additional co-collected materials in the sample sediment, such as mucus, fecal residue, food debris, or soil, may also contribute detectable parasite eDNA if there’s any. We aimed to investigate whether centrifuged sediment from ethanol-preserved gastropod samples could serve as a practical matrix for molecular detection. To improve screening efficiency, centrifuge-generated pellet sediment from each sample was allotted into pooled composites. Composite extracts were screened for A. cantonensis DNA using the AcanR3990 qPCR assay. Positive composite pools were then resolved through follow-up testing of individual constituent samples. In parallel, preservative ethanol supernatants were evaluated using FTA Micro Cards as an additional DNA capture method. In our experiment, the negative and positive controls we created performed as expected, demonstrating that the extraction and qPCR workflow could distinguish non-amplifying samples from spiked positive material. Composite qPCR screening identified multiple candidate positive pools in both microscopy-negative and previously uncharacterized samples, and follow-up individual testing recovered positive detections from several of those pools. However, conventional PCR and Sanger sequencing did not consistently confirm qPCR positives due to method limitations. Preliminary FTA card testing further suggested that the parasite-associated DNA can be recovered from preservative ethanol fractions in a field collection setup when lab equipment or facilities are not available. Together, this work develops and evaluates a scalable molecular method for environmental detection of A. cantonensis in gastropod preservation media. By focusing on non-destructive sampling of preservative ethanol and associated sediment, this approach has potential application not only for contemporary surveillance but also for retrospective analysis of archived specimens. More broadly, this study contributes to improved environmental surveillance of rat lungworm disease by introducing a more field-applicable and less destructive molecular detection of environmental-collected and preserved gastropods. This may help clarify whether ecological infection patterns in gastropod hosts align with, or diverge from, observed trends in reported human disease globally.