Detection of rat lungworm (Angiostrongylus cantonensis) infection by real-time PCR from the peripheral blood of animals: a preliminary study.

Rat lungworm disease or neuroangiostrongyliasis is a cerebral parasitic infection that affects humans and animals alike. Its clinical signs and symptoms can range from mild self-resolving to serious life-threatening conditions. Studies suggest therapeutic interventions during the early stages of infection to be more effective than in later stages. However, early diagnosis of infection is usually problematic without the knowledge of exposure and/or detection of the parasite's DNA or antibody against the parasite in the cerebrospinal fluid. This requires a lumbar puncture, which is an invasive procedure that generally requires hospitalization. This study evaluates an affordable and less invasive alternative to detect parasitic DNA by PCR from the peripheral blood of potentially infected animals. Blood samples from 58 animals (55 dogs and 3 cats) with clinical suspicion of infection were submitted to our lab between February 2019 and August 2022 by local, licensed veterinarians. DNA was extracted from whole blood, plasma, serum, and/or packed cells using the Qiagen DNeasy Blood & Tissue Kit as per the manufacturer's protocol. All 58 animals were tested by real-time PCR using the AcanITS1 assay and 32 of these animals (31dogs; 1 cat) were also tested using the AcanR3990 assay. The PCR results for both assays were classified into strongly positive > positive > weakly positive > negative, and equivocal for ambiguous results, based on the strength of the signal. The percent infection detected using the AcanITS1 and AcanR3990 assays was 12.72% (7/55) and 20.68% (6/29), respectively. The overall percent infection detected was 34.37% (11/32), with only two animals testing positive by both assays. The three cats involved in this study tested negative by both assays. These results are promising and warrant further investigations to increase sensitivity including variables that might affect detection in the blood, such as parasite load, and laboratory methodologies.

Rat lungworm (Angiostrongylus cantonensis) active larval emergence from deceased bubble pond snails (Bullastra lessoni) into water.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Clinical Efficacy and Safety of Albendazole and Other Benzimidazole Anthelmintics for Rat Lungworm Disease (Neuroangiostrongyliasis): A Systematic Analysis of Clinical Reports and Animal Studies.

The safety and efficacy of benzimidazole anthelmintics for the treatment of rat lungworm disease (neuroangiostrongyliasis) have been questioned regardless of numerous experimental animal studies and clinical reports. In this review, 40 of these experimental animal studies and 104 clinical reports are compiled with a focus on albendazole. Among the 144 articles involving an estimated 1034 patients and 2561 animals, 4.1% were inconclusive or vague regarding the use of benzimidazoles. Of the remaining 138 articles, 90.5% found benzimidazoles to be safe and effective (885 patients, 2530 animals), 4.3% as safe but ineffective (73 patients, 3 animals), and 5.0% caused adverse reactions (7 patients, 28 animals). Among those clinical reports that described a confirmed diagnosis of neuroangiostrongyliasis in which albendazole monotherapy was used, 100% reported high efficacy (743 patients, 479 animals). In those where albendazole-corticosteroid co-therapy was used, 97.87% reported it to be effective (323 patients, 130 animals).

Angiostrongylus cantonensis Nematode Invasion Pathway, Mallorca, Spain.

Neural angiostrongyliasis is an emerging zoonosis caused by the rat lungworm, Angiostrongylus cantonensis. In humans, infection with this nematode often results in eosinophilic meningitis and other severe disorders of the central nervous system. Europe was deemed a nonendemic region until 2018, when A. cantonensis worms were detected on the Mediterranean island of Mallorca, Spain, a tourism hotspot. Since that time, a sentinel surveillance system and a molecular approach have been used to follow the invasion path of the rat lungworm on the island. A. cantonensis worms have been found in animals from 8 locations on the island over 3 consecutive years. Our preliminary results show a recognizable pattern of clinical signs in infected hedgehogs and a single mitochondrial haplotype circulating in Mallorca. We present strong evidence confirming that the rat lungworm has successfully established and colonized an island in Europe and discuss observations and possible strategies for its early detection across continental Europe.

Evaluation of the mechanism of action of albendazole on adult rat lungworm (Angiostrongyluscantonensis).

Albendazole is considered the anthelmintic of choice for the management of rat lungworm disease (neuroangiostrongyliasis), due to its broad spectrum of nematocidal activity and its ability to cross the blood-brain barrier. Albendazole binds to ß-tubulins, preventing their polymerization into microtubules, thereby corrupting the cascade of cell division at metaphase, which ultimately leads to the death of individual cells and eventually the death of the parasite. Inhibition of microtubule formation will also hinder the axoplasmic transport system, affecting the neuronal activities of the parasite. While this mechanism has been explicated in other parasitic and non-parasitic nematodes, it has never been evaluated in Angiostrongylus cantonensis. This study evaluates the antimitotic effects of albendazole sulphoxide (active metabolite) on the microtubules of adult A. cantonensis using the tubulin polymerization assay and measures its effects on worm viability using the colorimetric MTT assay. Three different concentrations of albendazole (62.5 µM, 250 µΜ, and 1 mM) were evaluated. We saw a statistically significant dose-dependent reduction in the band intensity of polymerized tubulins (or microtubules) (P = 0.019), suggesting that albendazole imparts its antimitotic effect in a dose-dependent manner. Similarly, our MTT assay showed a dose-dependent decrease in formazan intensity (proportional to cell viability), suggesting that the rate of nematocidal activity of albendazole is also proportional to its concentration. In compiling the results from both these experiments, a correlation between the microtubule assembly and worm viability is evident.

A Hawaii public education programme for rat lungworm disease prevention.

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.

Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010-2020) and results for a novel, highly sensitive qPCR assay.

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.

In vitro comparison of treatments and commercially available solutions on mortality of Angiostrongylus cantonensis third-stage larvae.

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.

In vitro efficacy of anthelmintics on Angiostrongylus cantonensis L3 larvae.

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 (rat lungworm) in Florida, USA: current status.

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.

Demographics of the semi-slug Parmarion martensi, an intermediate host for Angiostrongylus cantonensis in Hawai'i, during laboratory rearing.

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).

Chronic neuroangiostrongyliasis: case study of chronic presentations in Hawaii.

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.

Modelling the distribution in Hawaii of Angiostrongylus cantonensis (rat lungworm) in its gastropod hosts.

Angiostrongylus cantonensis (rat lungworm), a parasitic nematode, is expanding its distribution. Human infection, known as angiostrongyliasis, may manifest as eosinophilic meningitis, an emerging infectious disease. The range and incidence of this disease are expanding throughout the tropics and subtropics. Recently, the Hawaiian Islands have experienced an increase in reported cases. This study addresses factors affecting the parasite's distribution and projects its potential future distribution, using Hawaii as a model for its global expansion. Specimens of 37 snail species from the Hawaiian Islands were screened for the parasite using PCR. It was present on five of the six largest islands. The data were used to generate habitat suitability models for A. cantonensis, based on temperature and precipitation, to predict its potential further spread within the archipelago. The best current climate model predicted suitable habitat on all islands, with greater suitability in regions with higher precipitation and temperatures. Projections under climate change (to 2100) indicated increased suitability in regions with estimated increased precipitation and temperatures, suitable habitat occurring increasingly at higher elevations. Analogously, climate change could facilitate the spread of A. cantonensis from its current tropical/subtropical range into more temperate regions of the world, as is beginning to be seen in the continental USA.

Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus).

Angiostrongylus 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.

Angiostrongylus cantonensis Eosinophilic Meningitis in an Infant, Tennessee, USA.

Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of eosinophilic meningoencephalitis worldwide. This parasite is endemic to Southeast Asia and the Pacific Islands, and its global distribution is increasing. We report A. cantonensis meningoencephalitis in a 12-month-old boy in Tennessee, USA, who had not traveled outside of southwestern Tennessee or northwestern Mississippi.

Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.

This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

An epidemiological study of A. cantonensis in Jamaica subsequent to an outbreak of human cases of eosinophilic meningitis in 2000.

The infection status of angiostrongylosis in Jamaica was assessed in wild rats and molluscs in the 5 years following the major outbreak of eosinophilic meningitis (EM) in 2000. Parasitological analyses of 297 Rattus rattus and 140 Rattus norvegicus, and 777 terrestrial molluscs from all 14 Parishes on the island revealed Angiostrongylus cantonensis in 32·0% of the rats and in 12·5% of the molluscs. Multivariate analyses confirmed that A. cantonensis occurred significantly more frequently in R. rattus (Odds Ratio [OR] = 1·76), while mean infection intensity in R. rattus was also significantly higher (16·8) than R. norvegicus (11·3) (Mann-Whitney U-test: P = 0·01). Third-stage larvae of A. cantonensis were detected in 29% of 86 Pleurodonte spp.; in 20% of five Poteria spp.; in 18·7% of 369 Thelidomus asper; in 11% of 18 Sagda spp.; and in 6% of 24 veronicellid slugs. Most rodent infections occurred in Northeastern Jamaica (OR = 11·66), a region where infected molluscs were also abundant. Given the prevalence of A. cantonensis infection in rats has significantly increased since the 2000 outbreak, and that a survey of human infections revealed at least ten autochthonous cases in the last 15 years, angiostrongylosis persists as an important zoonosis in Jamaica.

Geographic Range Expansion for Rat Lungworm in North America.

Using quantitative PCR analysis and DNA sequencing, we provide evidence for the presence of rat lungworm (Angiostrongylus cantonensis) in Oklahoma, USA, and identified a potentially novel rat host (Sigmodon hispidus). Our results indicate a geographic range expansion for this medically and ecologically relevant parasite in North America.

Local terrestrial snails as natural intermediate hosts of the zoonotic parasite Angiostrongylus cantonensis in the new European endemic area of Valencia, Spain.

AIM: The rat lungworm, Angiostrongylus cantonensis, has recently been found in the city of Valencia, parasitizing rats, Rattus norvegicus and Rattus rattus, its natural definitive hosts. This is the first finding of this zoonotic nematode in continental Europe. After informing local and national health authorities, the collection of local terrestrial snails took place with the aim of elucidating their potential role as intermediate hosts of A. cantonensis. METHODS AND RESULTS: A total of 145 terrestrial snails, belonging to the species Cernuella virgata, Cornu aspersum, Eobania vermiculata, Otala punctata, Pseudotachea splendida, Rumina decollata and Theba pisana, were randomly collected between May and December 2022 in public gardens, parks and orchards in six districts of Valencia, in five of which A. cantonensis had been reported previously in rats. Once collected and identified, the snails were frozen at -20°C. Subsequently, the DNA was isolated and screened by PCR using specific primers targeting the A. cantonensis COI gene. Seven individual snails, belonging to the species C. virgata, C. aspersum and T. pisana, were positive, for an overall prevalence of 4.8%. The PCR product from one of them was sequenced by Sanger sequencing. CONCLUSIONS: The three positive terrestrial snail species are among the edible species that are frequently included in various dishes in Spain. C. virgata is reported as a previously unrecorded intermediate host and should be added to the list of more than 200 species of terrestrial snails that have been reported worldwide as intermediate hosts of the rat lungworm. Considering that these terrestrial snails may release infective larvae of A. cantonensis on leafy green vegetables on which they feed and during their handling and preparation for consumption, prophylactic measures to prevent human neuroangiostrongyliasis in Valencia and other regions to which this zoonotic parasite may spread are recommended.