Epidemiological, clinical and laboratory aspects of Angiostrongylus cantonensis infection: an integrative review.

This integrative literature review study analyzes the findings of the last 5 years of the parasite Angiostrongylus cantonensis. It is known that this nematode is found in the pulmonary arteries of rats, where it remains as a definitive host. From mice, the cycle spreads to animals, such as snails, which in contact with humans can trigger the disease. In humans, the parasite causes several neurological, abdominal manifestations and mainly meningitis. Based on the review of studies, its epidemiology shows worldwide distribution, although there are endemic cases for this parasite, such as asian countries. Laboratory findings generally showed altered CSF with turbidity, increased protein and eosinophilia, which generated meningeal signs in the patient, moreover MRI exams showed multiple alterations. Rare findings of the nematode in the eyeball, lung and signs of peritoneal inflammation were reported, which requires further studies to understand the whole pathophysiology. Finally, conservative treatment based on anthelmintics and anti-inflammatories brought good responses, although there are reports of deaths, which demonstrates the importance in the prevention and therapy of this disease.

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.

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis.

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood-brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-ß. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.

Eosinophilic meningitis in New Caledonia: The role of Angiostrongylus cantonensis?

INTRODUCTION: Eosinophilic meningitis is a rare form of meningitis with sequelae or death occurring in approximately 2-3% of cases. The most frequent etiological agent is the parasite Angiostrongylus cantonensis. The aim of this study was to characterize New Caledonian cases and to assess the extent to which of A. cantonensis was involved. MATERIAL AND METHODS: We performed a retrospective study of all cases of eosinophilic meningitis (EM) admitted to the Territorial Hospital of New Caledonia, from 2004 to 2019. We performed a descriptive and a multivariate analysis to identify association of variables with severe and fatal cases (or cases with sequelae). CONCLUSION: Angiostrongyliasis was confirmed as being responsible for 17 of the 92 reported EM cases in New Caledonia from 2004 to 2019 with most being young adults and non-walking infants, and with two peaks of incidence one during the dry season and one during the rainy season. Considering the high incidence and regularity of cases, the potential reservoirs should be identified to target prevention campaigns.

Low genetic diversity and the phylogeny of Achatina fulica, an intermediate host of Angiostrongylus cantonensis in Thailand, inferred from 16S mitochondrial sequences.

The giant African land snail, Achatina fulica, is a pulmonate land snail of the Achatinidae family. The snail is native of East Africa and has spread to tropical and subtropical areas in different parts of the world. Achatina fulica is an important agricultural pests and the intermediate host for Angiostrongylus spp., especially Angiostrongylus cantonensis, which causes eosinophilic meningoencephalitis in humans. However, information on the genetic diversity and phylogenetic relationships among A. fulica populations in Thailand is limited. The goal of this study was to evaluate the genetic diversity and analyze haplotype network relationships between A. fulica populations in Thailand and other areas of the world. We collected A. fulica from 22 provinces in 6 regions of Thailand and analyzed a phylogenetic tree and haplotype network based on a fragment of 239 base pairs of the 16S rRNA sequence. The maximum likelihood, neighbor joining, and Bayesian inference trees for the 133 A. fulica 16S rRNA sequences revealed only one group, closely related only to A. fulica haplotype C, in all regions of Thailand. The haplotype network analysis showed that haplotype C is associated with F, P, E, Q, H and D. The genetic distance between the haplotype and A. fulica isolates in Thailand varies from 0 and 0.024, with a total of 21 variable sites. Haplotype C is predominant in Thailand and associated with other haplotypes from several areas. As inferred from 16S rDNA sequences, this study demonstrated low genetic diversity in A. fulica in Thailand. Low genetic diversity in the population can increase susceptibility to A. cantonensis infection, which may be associated with the current distribution of A. cantonensis.

Alternative pathways in Angiostrongylus cantonensis (Metastrongyloidea: Angiostrongylidae) transmission.

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.

Pathology of Angiostrongylus cantonensis infection in two model avian hosts.

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.

Gross, microscopic, radiologic, echocardiographic and haematological findings in rats experimentally infected with Angiostrongylus cantonensis.

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.

Dexamethasone downregulates the expressions of MMP-9 and oxidative stress in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

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.

Magnetic resonance imaging in dogs with neuroangiostrongyliasis (rat lungworm disease).

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.

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.

Variation in Angiostrongylus cantonensis infection in definitive and intermediate hosts in Hawaii, a global hotspot of rat lungworm disease.

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.

Guidelines for the diagnosis and treatment of neuroangiostrongyliasis: updated recommendations.

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.

Angiostrongylus cantonensis infection of non-native rats in Mount Makiling Forest Reserve, the Philippines.

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.

Mitochondrial dynamics in Angiostrongylus cantonensis-infected mouse brain.

Angiostrongylus cantonensis is one of the most widespread parasites causing central nervous system (CNS) diseases in mammals. Since the mitochondrion is an essential cell organelle responsible for both physiological and pathological processes, its dysfunction might lead to inflammation and multiple disorders. In this study we aimed to investigate the changes in mitochondrial dynamics that occur in the mouse brain upon infection with A. cantonensis, using molecular biology techniques such as polymerase chain reaction (PCR), western blot analysis, transmission electron microscopy (TEM), and different staining methods. Here, we show that mouse brain infected with A. cantonensis exhibits altered mitochondrial dynamics, including fission, fusion, and biogenesis. Additionally, we demonstrate that caspases and B-cell lymphoma 2 (BCL-2) were significantly upregulated in A. cantonensis-infected brain. These results are indicative of the occurrence of apoptosis during A. cantonensis infection, which was further confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These findings suggest the change in mitochondrial dynamics in A. cantonensis-infected brain, providing another point of view on the pathogenesis of meningoencephalitis caused by A. cantonensis infection.

Angiostrongylus cantonensis infection induces MMP-9 and causes tight junction protein disruption associated with Purkinje cell degeneration.

Angiostrongylus cantonensis causes a human central nervous system (CNS) infection characterized by eosinophilic meningitis or meningoencephalitis. Individuals infected with A. cantonensis exhibit unbalanced walking. The mechanism of extensive neurological impairments of hosts caused by A. cantonensis larvae remains unclear. Tight junction proteins (e.g., claudin-5 and zonula occludens-1) are the most important regulators of paracellular permeability and cellular adhesion. In a previous study, we found that increased matrix metalloproteinase-9 (MMP-9) activity may be associated with blood-CNS barrier disruption and/or the degeneration of Purkinje cells in eosinophilic meningitis caused by A. cantonensis. In the present study, the co-localization of MMP-9 and tight junction proteins on the degeneration of Purkinje cells was measured via confocal laser scanning immunofluorescence microscopy. The statistical evidence indicated that MMP-9 correlated between tight junction protein disruption and Purkinje cell degeneration at 20 days post-infection with A. cantonensis. In conclusion, Purkinje cell degeneration is highly correlated with tight junction protein disruption via the MMP-9 activation pathway.

Mollusk microbiota shift during Angiostrongylus cantonensis infection in the freshwater snail Biomphalaria glabrata and the terrestrial slug Phillocaulis soleiformis.

In the present work, we reported for the first time the microbiome from Phyllocaulis soleiformis and Biomphalaria glabrata assessed using high-throughput DNA sequencing pre- and post-infection with the helminth parasite Angiostrongylus cantonensis. B. glabrata and P. soleiformis were experimentally infected with A. cantonensis. Fecal DNAs from control and infected groups were extracted and subjected to 16S rRNA high-throughput sequencing survey. No significant differences were found in the alpha diversity indexes in Phyllocaulis and Biomphalaria experiments independently. PCoA analysis using the unweighted UniFrac measures showed that both microbiotas behaved differently depending on the host. In Biomphalaria microbiota, control and infected groups were significantly different (p = 0.0219), while Phyllocaulis samples were not (p = 0.5190). The microbiome of P. soleiformis infected with A. cantonensis showed a significant decrease of Sphingobacterium and a substantial increase of Cellvibrio when compared to a control group. The microbiome of B. glabrata infected with A. cantonensis showed a significant decline in the abundance of Flavobacterium, Fluviicola, Nitrospira, Vogesella and an OTU belonging to the family Comamonadaceae, and a significant increase of Uliginosibacterium and an OTU belonging to the family Weeksellaceae when compared to a control group. Overall, the microbiome data reported here provided valuable information with regard to the diversity of bacterial communities that comprise the gut microbiome of gastropods. Furthermore, we report here the effect of the infection of the helminth A. cantonensis in the ratio and distribution of the fecal microbiome of the snails. Further studies are highly valuable in order to better understand those interactions by comparing different microbiome profiles and mollusk models. By now, we anticipate that ecological studies will take significant advantage of these advances, particularly concerning improving our understanding of helminth-microbiome-host interactions.

Physiology and immunity of the invasive giant African snail, Achatina (Lissachatina) fulica, intermediate host of Angiostrongylus cantonensis.

As one of the most successful invasive land snail species, Achatina (Lissachatina) fulica Bowdich, 1822 has achieved wide global distribution, particularly in (sub)tropical regions, with further dispersal likely due to climate change. This species of giant African snails (up to 17 cm shell length) is a pest that has extensive negative impact on agriculture and can serve as vector for several parasites, including Angiostrongylus cantonensis, a nematode parasite that causes (human) eosinophilic meningitis, an emergent disease. Investigation showed that A. cantonensis infection negatively impacts the metabolism of A. fulica by depleting polysaccharide stores of the intermediate host, compromising the energy balance of the snail. A review of the literature indicates that A. fulica possesses potent innate type immune defenses to counter infection, including phagocytic hemocytes capable of deploying reactive oxygen species and lectins for non-self recognition, a serine protease-dependent coagulation response (not observed in other taxa of gastropods), as well as antimicrobial proteins including achacin, an antimicrobial protein. A recent chromosome level genome assembly will facilitate progressively detailed characterization of these immune features of A. fulica. We strongly encourage further immunological studies of A. fulica, ranging from organismal level to molecular biology to gain better understanding of the A. fulica internal defense response to nematode pathogens like A. cantonensis and the contribution of immune function to the invasiveness of (snail) species. Characterization of immunity of A. fulica, representing the understudied Stylommatophora (panpulmonate landsnails) will also broaden the comparative immunology of Gastropoda.