Angiostrongylus cantonensis (rat lungworm) is a tropical and subtropical parasitic nematode, with infections in humans causing angiostrongyliasis (rat lungworm disease), characterized by eosinophilic meningitis. Hawaii has been identified as a global hotspot of infection, with recent reports of high infection rates in humans, as well as rat definitive and snail intermediate hosts. This study investigated variation in A. cantonensis infection, both prevalence and intensity, in wild populations of two species of rats (Rattus exulans and R. rattus) and one species of snail (Parmarion martensi). An overall infection prevalence of 86.2% was observed in P. martensi and 63.8% in rats, with R. exulans (77.4%) greater than R. rattus (47.6%). We found infections to vary with environmental and host-related factors. Body mass was a strong predictor of infection in all three species, with different patterns seen between sexes and species of rats. Infection prevalence and intensity for R. exulans were high in May 2018 and again in February 2019, but generally lower and more variable during the intervening months. Information on sources of variability of infection in wild host populations will be a crucial component in predicting the effectiveness of future disease surveillance or targeted management strategies.

A number of studies had been conducted on rat species diversity in the Philippines, however, there is a dearth of information on the extent of Angiostrongylus spp. infection in various ecological niches. Collection of non-native rat samples occurred in various sampling sites categorized as residential, agricultural and agro-forest in Philippine Mount Makiling Forest Reserve (MMFR) and its adjacent areas . Three species of non-native rats were captured including Rattus tanezumi, Rattus norvegicus and Rattus exulans. Of the total 90 non-native rats collected, 24.4% were found infected with Angiostrongylus cantonensis. Statistical analysis showed significant difference among rat species with highest prevalence observed in R. exulans (42.9%) followed by R. tanezumi (29.8%) and R. norvegicus (7.7%) (P = 0.047). Angiostrongylus cantonensis infection was significantly higher in adults (29.4%) than in juvenile rats (9.1%) (P = 0.050). However, results showed no significant difference in A. cantonensis infection between male (43.3%) and female (18.3%) rats and between wet (31.6%) and dry seasons (19.2%) (P > 0.05). Moreover, this study also revealed that rats from agricultural and agro-forest areas showed significantly higher prevalence than residential areas. With urbanization and the everchanging landscape of MMFR watersheds and buffer zones, zoonotic transmission can pose health threats to the local people.

Angiostrongylus cantonensis has been found in Florida, USA, from the panhandle in the north to Miami and surrounding areas in the southern parts of the state, in both definitive and intermediate hosts in a limited studies completed in 2015. Additional studies have identified this parasite in a variety of intermediate hosts, both native and non-native gastropod species, with new host species recorded. Many areas in Florida with higher A. cantonensis prevalence were those with a high human population density, which suggests it is a matter of time before human infections occur in Florida. Case reports in the state currently involve non-human primates and include a gibbon and orangutan in Miami. Here, we report the current status of A. cantonensis in the state, as well as the infection in a capuchin monkey and presumptive infection in a red ruffed lemur in Gainesville, Florida.

The semi-slug, Parmarion martensi, is an intermediate host of the zoonotic nematode, Angiostrongylus cantonensis, the aetiological agent of neuroangiostrongyliasis or rat lungworm disease in humans. Rearing methods were developed for P. martensi to facilitate studies on nematode transmission and control. Parmarion martensi exhibited high survivorship when reared on a diet of dog food and fresh fruits and vegetables in temperature-controlled cabinets at 21.4°C, 98% relative humidity and 12:12 L:D cycle. Rearing containers were lined with moist paper towels for substrate and plastic pots were provided for hiding/resting and egg-laying. Under these conditions, time to first reproduction was 165.3 ± 12.3 days, fecundity was approximately 34.5 ± 7.8 eggs per adult, and hatch rate was 52.7 ± 3.2%. Survivorship post egg hatch was 86.2 ± 2.9% at 30 days (neonates had a mortality rate of about 14%) and 99% thereafter for up to a year. The demographics of laboratory-reared and wild-caught P. martensi were similar except for the weight of reproductive adults, which was significantly higher in laboratory-reared adults (4.0 ± 0.2 g) than in field-collected adults (1.5 ± 0.1 g).

Although the gross and microscopic pathology in rats infected with Angiostrongylus cantonensis has been well described, corresponding changes detected using diagnostic imaging modalities have not been reported. This work describes the cardiopulmonary changes in mature Wistar rats chronically infected with moderate burdens of A. cantonensis using radiology, computed tomography (CT), CT angiography, echocardiography, necropsy and histological examinations. Haematology and coagulation studies were also performed. Thoracic radiography, CT and CT angiography showed moderately severe alveolar pulmonary patterns mainly affecting caudal portions of the caudal lung lobes and associated dilatation of the caudal lobar pulmonary arteries. Presumptive worm profiles could be detected using echocardiography, with worms seen in the right ventricular outflow tract or straddling either the pulmonary and/or the tricuspid valves. Extensive, multifocal, coalescing dark areas and multiple pale foci affecting the caudal lung lobes were observed at necropsy. Histologically, these were composed of numerous large, confluent granulomas and fibrotic nodules. Adult worms were found predominantly in the mid- to distal pulmonary arteries. An inflammatory leukogram, hyperproteinaemia and hyperfibrinogenaemia were found in most rats. These findings provide a comparative model for A. cantonensis in its accidental hosts, such as humans and dogs. In addition, the pathological and imaging changes are comparable to those seen in dogs infected with Angiostrongylus vasorum, suggesting rats infected with A. cantonensis could be a model for dogs with A. vasorum infection.

In order to elucidate the infection pathways of third stage larvae (L3) of Angiostrongylus cantonensis, we performed experiments to assess: (i) the shedding of L3 from two species of experimental veronicellid slugs drowned in water and the ratio of emerged larvae, (ii) the transmission of viable L3 from drowned terrestrial gastropods to aquatic snails, and (iii) the transmission of viable L3 between terrestrial snails. Molluscs were experimentally infected by first stage larvae (L1) of A. cantonensis. Significantly more L3 larvae were released from Veronicella cubensis than from Veronicella sloanei. Numerous L3 were observed in the muscular foot, and also in the connective tissue between internal organs. Experimental exposure of P. maculata to L3 of A. cantonensis liberated from other gastropod species led to their infection and the infectivity of larvae after intermediesis was demonstrated by infection of laboratory rats (Rattus norvegicus). The transmission of L3 was observed in three out of four experiment replications and L3 were retrieved from 6 out of 24 Subulina octona snails. The infected synanthropic molluscs represent a key component in the epidemiology of human infections by A. cantonensis. Escape of L3 larvae from bodies of dead snails or slugs and their ability to infect further gastropod hosts (intermediesis) represents a public health risk. Thus, control of molluscs living in peri-domestic environment is an essential part of prevention of human infections.

Angiostrongylus cantonensis causes severe neurological disorders in a wide range of warm-blooded animals, including several avian species. A laboratory isolate of A. cantonensis originating from French Polynesia, genotyped as clade 2, was used to assess the effect of experimental infection in chicken and Japanese quail. Low dose groups of birds were infected orally by 100 L3 larvae, high dose groups by 1500 L3 larvae and the birds in the third group were fed three infected snails, mimicking a natural infection. Clinical signs during the first week after infection, haematology, biochemistry, gross lesions and histology findings were used to assess the pathology of the infection. Some of the infected birds showed peripheral eosinophilia, while mild neurological signs were seen in others. No larvae were observed in serial sections of the central nervous system of infected birds 1 week after infection and no major gross lesions were observed during necropsy; histopathology did not reveal lesions directly attributable to A. cantonensis infection. Our results suggest that galliform birds are not highly susceptible to A. cantonensis infection and open a question of the importance of Galliformes in endemic areas as natural pest control, lowering the number of hosts carrying the infective larvae.

The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells μL−1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, CT values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.

Steroids have been shown to be beneficial in patients and mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection; however, the mechanism for this beneficial effect is unknown. We speculated that the effect of steroids in eosinophilic meningitis caused by A. cantonensis infection may be mediated by the downregulation of matrix metallopeptidase-9 (MMP-9) and oxidative stress pathways via glucocorticoid receptors (GRs). We found blood–brain barrier (BBB) dysfunction in mice with eosinophilic meningitis 2–3 weeks after infection as evidenced by increased extravasation of Evans blue and cerebrospinal fluid (CSF) albumin levels. The administration of dexamethasone significantly decreased the amount of Evans blue and CSF albumin. The effect of dexamethasone was mediated by GRs and heat shock protein 70, resulting in subsequent decreases in the expressions of nuclear factor kappa B (NF-κB), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in the CSF and brain parenchymal after 2 weeks of steroid administration. Steroid treatment also decreased CSF/brain homogenate MMP-9 concentrations, but had no effect on CSF MMP-2 levels, indicating that MMP-9 rather than MMP-2 played a major role in BBB dysfunction in mice with eosinophilic meningitis. The concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) gradually increased after 1–3 weeks of infection, and the administration of dexamethasone significantly downregulated the concentration of oxidized derivative 8-OHdG in CSF. In conclusion, increased 8-OHdG and MMP-9 concentrations were found in mice with eosinophilic meningitis caused by A. cantonensis infection. The effect of dexamethasone was mediated by GRs and significantly decreased not only the levels of 8-OHdG and MMP-9 but also NF-κB, JNK and ERK.

The magnetic resonance imaging (MRI) appearance of the brain and spinal cord in humans with neuroangiostrongyliasis (NA) due to Angiostrongylus cantonensis infection has been well reported. Equivalent studies in animals are lacking. This case series describes clinical and MRI findings in 11 dogs with presumptively or definitively diagnosed NA. MRI of the brain and/or spinal cord was performed using high-field (1.5 T) or low-field (0.25 T) scanners using various combinations of transverse, sagittal, dorsal and three-dimensional (3D) T1-weighted (T1W), transverse, sagittal and dorsal T2-weighted (T2W), T2W fluid-attenuated inversion recovery (FLAIR) and T2*-weighted (T2*W) gradient echo (GRE), dorsal T2W short tau inversion recovery (STIR) and post-gadolinium transverse, sagittal, dorsal and 3D T1W and transverse T2W FLAIR sequences. In 4/6 cases where the brain was imaged, changes consistent with diffuse meningoencephalitis were observed. Evidence of meningeal involvement was evident even when not clinically apparent. The spinal cord was imaged in 9 dogs, with evidence of meningitis and myelitis detected in regions consistent with the observed neuroanatomical localization. Pathognomonic changes of neural larva migrans, as described in some human patients with NA, were not detected. NA should be considered in the differential diagnosis of dogs with MRI evidence of focal or diffuse meningitis, myelitis and/or encephalitis, especially in areas where A. cantonensis is endemic. If not precluded by imaging findings suggestive of brain herniation, cerebrospinal fluid (CSF) collection for cytology, fluid analysis, real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) testing should be considered mandatory in such cases after the MRI studies.

Education is essential for lowering cases of Angiostrongyliasis in Hawaii. A professional development course on rat lungworm disease (RLWD) prevention was offered to Hawaii K-12 teachers during the 2018–2019 school year. Fourteen teachers, 11 from Hawaii Island and three from Maui, representing grades K, 2, 6–8, and 10−12, completed workshops and activities and received course credit. Teachers reached 652 students and educated 86 teachers and staff and approximately 900 community members including parents. A pre-course survey showed 55% of teachers agreed to a basic understanding of rat lungworm and its impact on Hawaii; this increased to 100% post-course. A sixth-grade class was the first to document the arrival of a highly competent intermediate host of the rat lungworm in a new location, an important discovery that initiated community awareness and control efforts. Six, 1-day workshops were held in 2018–2019, each on one of the main Hawaiian Islands. These were attended by 106 participants including teachers, community educators, and interested individuals from agencies, non-profits, businesses and the private sector. Of participants surveyed, 100% responded the workshop improved their overall understanding of RLWD. Efforts are being made to continue these programmes.

On Hawai‘i Island, an increase in human neuroangiostrongyliasis cases has been primarily associated with the accidental ingestion of Angiostrongylus cantonensis L3 in snails or slugs, or potentially, from larvae left behind in the slug's slime or feces. We evaluated more than 40 different treatments in vitro for their ability to kill A. cantonensis larvae with the goal of identifying a safe and effective fruit and vegetable wash in order to reduce the risk of exposure. Our evaluation of treatment lethality was carried out in two phases; initially using motility as an indicator of larval survival after treatment, followed by the development and application of a propidium iodide staining assay to document larval mortality. Treatments tested included common household products, consumer vegetable washes and agricultural crop washes. We found minimal larvicidal efficacy among consumer-grade fruit and vegetable washes, nor among botanical extracts such as those from ginger or garlic, nor acid solutions such as vinegar. Alkaline solutions, on the other hand, as well as oxidizers such as bleach and chlorine dioxide, did show larvicidal potential. Surfactants, a frequent ingredient in detergents that lowers surface tension, had variable results, but dodecylbenzene sulfonic acid as a 70% w/w solution in 2-propanol was very effective, both in terms of the speed and the thoroughness with which it killed A. cantonensis L3 nematodes. Thus, our results suggest promising directions for future investigation.

This paper describes chronic features of neuroangiostrongyliasis (NAS), a long-term outcome of the disease that has not been adequately described. Current and past literature is predominantly limited to acute manifestations of NAS, and mention of chronic, ongoing clinical symptoms is usually limited to brief notes in a discussion of severe cases. This study investigated the long-term outcomes in ten individuals who were diagnosed with acute neuroangiostrongyliasis in Hawaii between 2009 and 2017. The study demonstrates a significant number of persons in Hawaii sustain residual symptoms for many years, including troublesome sensory paresthesia (abnormal spontaneous sensations of skin experienced as ‘burning, pricking, pins and needles’; also described as allodynia or hyperesthesia) and extremity muscle pains. As a consequence, employment and economic hardships, domestic relocations, and psychological impairments affecting personal relationships occurred. The study summarizes common features of chronic disease, sensory paresthesia and hyperesthesia, diffuse muscular pain, insomnia, and accompanying emotional distress; highlights the frequently unsuccessful endeavours of individuals struggling to find effective treatment; proposes pathogenic mechanisms responsible for prolonged illness including possible reasons for differences in disease presentation in Hawaii compared to Southeast Asia.

A subcommittee of the Hawaii Governor's Joint Task Force on Rat Lungworm Disease developed preliminary guidelines for the diagnosis and treatment of neuroangiostrongyliasis (NAS) in 2018 (Guidelines, 2018). This paper reviews the main points of those guidelines and provides updates in areas where our understanding of the disease has increased. The diagnosis of NAS is described, including confirmation of infection by real-time polymerase chain reaction (RTi-PCR) to detect parasite DNA in the central nervous system (CNS). The treatment literature is reviewed with recommendations for the use of corticosteroids and the anthelminthic drug albendazole. Long-term sequelae of NAS are discussed and recommendations for future research are proposed.

Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10–15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present ‘AcDIGFAAg’ enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.

Angiostrongylus cantonensis is the leading cause of eosinophilic meningitis worldwide, with life-threatening complications if not managed correctly. Previous in vitro studies have utilized change in motility patterns of adult female worms to assess the efficacy of anthelmintics qualitatively. However, it is the third stage larvae (L3) that are infectious to humans. With differential staining using propidium iodide penetration as the indicator of death, we can distinguish between dead and live larvae. This assay has enabled us to quantify the in vitro efficacy of nine clinically established anthelmintics on A. cantonensis L3. All drugs were tested at a 1 mm concentration. Piperazine and niclosamide were ineffective in inducing larval death; however, albendazole sulfoxide, pyrantel pamoate, diethylcarbamazine, levamisole and praziquantel were effective as compared to unexposed controls (P < 0.05). Ivermectin and moxidectin did not induce significant levels of mortality, but they considerably reduced larval motility almost immediately. This study indicates the need for further in vivo studies to determine the optimal dose and time frame for post-infection treatment with anthelmintics that demonstrated efficacy.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai‘i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies μL−1 to ~1 copy μL−1. All three assays consistently detected 50–100 copies μL−1 in triplicate and qPCR was able to detect ~13 copies μL−1 in triplicate. RPA-EXO was able to detect 25 copies μL−1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL−1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.