BACKGROUND: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. METHODS: In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. RESULTS: The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. CONCLUSION: These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Department of Botany and Zoology Faculty of Science Masaryk University Brno Czech Republic

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees Faculty of Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Czech Republic

Department of Pathology and Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Czech Republic

Department of Pharmaceutical Sciences College of Pharmacy University of Hawaii at Hilo Hilo Hiwaii USA

Institute of Parasitology Biology Centre Czech Academy of Sciences Ceske Budejovice Czech Republic

Laboratory of Parasitic Diseases National Institute of Allergy and Infectious Diseases Bethesda Maryland USA

Laboratory of Veterinary Parasitology Sydney School of Veterinary Science Faculty of Science University of Sydney Sydney New South Wales Australia

Parasitic Disease Branch Division of Parasitic Diseases and Malaria Center for Global Health Centers for Disease Control and Prevention Atlanta Georgia USA

RML Genomics Unit Research Technology Branch National Institute of Allergy and Infectious Diseases National Institutes of Health Hamilton Montana USA

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