Background Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3 rd 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 3 rd 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 five 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 A. 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.

Leinbach, Israel L.; Klasner, Ina H.; Kaluna, Lisa M.; Snook, Kirsten A.; Howe, M. Kathleen; Jacquier, Steven H.; Lange, Ingo; Atkinson, Abigail L.; Deane, Ashley R.; Niebuhr, Chris N.; and Siers, Shane R., "Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus)" (2019). AbstractAngiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Neuroangiostrongyliasis, caused by Angiostrongylus cantonensis , has been reported in Hawaiʻi since the 1950's. An increase in cases is being reported primarily from East Hawaiʻi Island, correlated with the introduction of the semi-slug Parmarion martensi . Households in areas lacking infrastructure for water must use rainwater catchment as their primary domestic water supply, for which there is no federal, state, or county regulation. Despite evidence that slugs and snails can contaminate water and cause infection, regulatory bodies have not addressed this potential transmission route. This study evaluates: 1) the emergence of live, infective-stage A . cantonensis larvae from drowned, non-native, pestiforous gastropods; 2) larvae location in an undisturbed water column; 3) longevity of free-living larvae in water; and 4) effectiveness of rainwater catchment filters in blocking infective-stage larvae. Larvae were shed from minced and whole gastropods drowned in either municipal water or rainwater with ~94% of larvae recovered from the bottom of the water column 72–96 hours post drowning. Infective-stage larvae were active for 21 days in municipal water. Histological sectioning of P . martensi showed proximity of nematode larvae to the body wall of the gastropod, consistent with the potential for shedding of larvae in slime. Gastropod tissue squashes showed effectivity as a quick screening method. Live, infective-stage larvae were able to traverse rainwater catchment polypropylene sediment filters of 20 μm, 10 μm, 5 μm, and 1 μm filtration ratings, but not a 5 μm carbon block filter. These results demonstrate that live, infective-stage A . cantonensis larvae emerge from drowned snails and slugs, survive for extended periods of time in water, and may be able to enter a catchment user's household water supply. This study illustrates the need to better investigate and understand the potential role of contaminated water as a transmission route for neuroangiostrongyliasis.

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16 Neuroangiostrongyliasis, caused by Angiostrongylus cantonensis, has been reported in Hawaiʻi 17 since the 1950's. An increase in cases is being reported primarily from East Hawaiʻi Island, 18 correlated with the introduction of the semi-slug Parmarion martensi. Households in areas 19 lacking infrastructure for water must use rainwater catchment as their primary domestic water 20 supply, for which there is no federal, state, or county regulation. Despite evidence that 21 contaminated water can cause infection, regulatory bodies have not addressed this potential 22 transmission route. This study evaluates: 1) the emergence of live, infective-stage A. cantonensis 23 larvae from drowned, non-native, pestiforous gastropods; 2) larvae location in an undisturbed 24 water column; 3) longevity of free-living larvae in water; and 4) effectiveness of rainwater 25 catchment filters in blocking infective-stage larvae. Larvae were shed from minced and whole 26 gastropods drowned in either municipal water or rainwater with >94% of larvae recovered from 27 the bottom of the water column. Infective-stage larvae were active for 21 days in municipal 28 water. Histological sectioning of P. martensi showed proximity of nematode larvae to the body 29 wall of the gastropod, consistent with the potential for shedding of larvae in slime. Gastropod 2 30 tissue squashes showed effectivity as a quick screening method. Live, infective-stage larvae were 31 able to traverse rainwater catchment polypropylene sediment filters of 20 µm, 10 µm, 5 µm, and 32 1 µm filtration ratings, but not a 5 µm carbon block filter. These results demonstrate that live, 33 infective-stage A. cantonensis larvae can and do emerge from drowned snails and slugs, survive 34 for extended periods of time in water, and that the potential exists that they enter the household 35 water supply. This study illustrates the need to better investigate and understand the potential 36 role of contaminated water as a transmission route for neuroangiostrongyliasis. 37 38 Introduction 39 The nematode Angiostrongylus cantonensis is established throughout the main Hawaiian Islands 40 with the possible exception of Lānaʻi [1, 2, 3]. The complex lifecycle of this parasite has been 41 well-described in the literature [4, 5, 6, 7]. In Hawaiʻi, Rattus rattus and Rattus exulans are 42 important definitive hosts, and many gastropod species are effective intermediate hosts including 43 Achatina fulica, Euglandia rosea, Laevicaulis alte, Limax maximus, Parmarion martensi and 44 Veronicella cubensis [1, 2, 8]. The third stage larva (L3) is harbored in the intermediate host, and45 it is this larval stage that is infective to rats and accidental hosts, including humans, as the L3 46 larvae can safely pass through the acidic environment of the mammalian gut. There are also 47 paratenic hosts that can carry the infective stage larvae; these include shrimp, prawns, crabs, 48 frogs, water monitor lizards, centipedes, and some planarians [7. 9, 10, 11, 12]. Of planarians, 49 the predacious Platydemou...