The recent introduction of Angiostrongylus cantonensis and its intermediate host Achatina fulica into Guadeloupe detected by phylogenetic analyses.

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is the main pathogen responsible for eosinophilic meningitis in humans. One of its intermediate snail hosts, Achatina fulica, was already present in many countries around the world before it appeared in the West Indies in the late 1980s. In the French territories in the Caribbean and northern South America, the first cases of human neuroangiostrongyliasis were reported in Martinique, Guadeloupe and French Guiana in 2002, 2013 and 2017, respectively. In order to better characterize angiostrongyliasis in Guadeloupe, particularly its geographical origin and route of introduction, we undertook molecular characterization of adult worms of Angiostrongylus cantonensis and its intermediate host Achatina fulica. METHODS: Genomic DNA of adult Angiostrongylus cantonensis and Achatina fulica was extracted and amplified by polymerase chain reaction (PCR) targeting the mitochondrial genes cytochrome B and C for A. cantonensis and 16S ribosomal RNA for A. fulica. The PCR products were sequenced and studied by phylogenetic analysis. RESULTS: Cytochrome B and cytochrome C molecular markers indicate a monophyletic lineage of A. cantonensis adult worms in Guadeloupe. Two sequences of A. fulica were identified. CONCLUSIONS: These results confirm the recent introduction of both Angiostrongylus cantonensis and Achatina fulica into Guadeloupe. Achatina fulica in Guadeloupe shares a common origin with those in Barbados and New Caledonia, while Angiostrongylus cantonensis in Guadeloupe shares a common origin with those in Brazil, Hawaii and Japan.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

[Prevalence of Angiostrongylus cantonensis infection in snails in Fujian Province from 2017 to 2021].

OBJECTIVE: To investigate the prevalence of Angiostrongylus cantonensis infection in market-sold and field-captured snails in Fujian Province, so as to provide the scientific basis for the formulation of the angiostrongyliasis control measures. METHODS: In each month from May to October during the period from 2017 through 2021, Pomacea snails were collected from two field fixed surveillance sites and Bellamya aeruginosa collected from one agricultural product market in Fuzhou City, while Pomacea and B. aeruginosa snails were collected from two agricultural product markets and four restaurants in Xiamen City. At least 50 Pomacea snails and 500 g B. aeruginosa were sampled each time. A. cantonensis infection was detected in Pomacea snails using lung microscopy, and in B. aeruginosa using a tissue homogenate method. RESULTS: A total of 9 531 Pomacea snails were detected for A. cantonensis infection in two cities of Fuzhou and Xiamen, and the overall prevalence of A. cantonensis infection was 4.40%, with the highest prevalence in 2017 (6.82%, 116/1 701) and the lowest prevalence in 2019 (3.46%, 83/2 400). The prevalence of A. cantonensis infection was significantly higher in Pomacea snails sampled from Fuzhou City (11.23%, 326/2 903) than from Xiamen City (1.40%, 93/6 628) (χ2 = 461.48, P < 0.01). A. cantonensis larvae were detected in larval Pomacea snails in two cities of Fuzhou and Xiamen each month. The prevalence of A. cantonensis infection in Pomacea snails appeared an overall tendency towards a rise with month in Fuzhou City, with the highest prevalence in October (15.24%), and there was a significant difference among month (χ2 = 14.56, P < 0.05), while the prevalence of A. cantonensis infection in Pomacea snails was low in Pomacea snails sampled from Xiamen City each month, with the highest prevalence in June (2.64%), and there was a significant difference among month (χ2 = 23.17, P < 0.05). A total of 18 966 B. aeruginosa snails were detected for A. cantonensis infection in two cities of Fuzhou and Xiamen, and the overall prevalence of A. cantonensis infection was 0.01%. CONCLUSIONS: A. cantonensis infection was identified in Pomacea and B. aeruginosa snails in Fujian Province from 2017 to 2021, and there is a potential risk of human A. cantonensis infection.

[Progress of researches on techniques for detection of Angiostrongylus cantonensis in intermediate host snails].

Angiostrongyliasis cantonensis is an emerging infectious disease in China. Snails are intermediate hosts of Angiostrongylus cantonensis and play a critical role in the transmission of angiostrongyliasis cantonensis. Detection of A. cantonensis in snails is an important part of epidemiological surveys. Currently, the rapid developments in the techniques for detection of A. cantonensis in snails facilitate the surveillance of angiostrongyliasis cantonensis and provide an important support for angiostrongyliasis cantonensis prevention and control. This review summarizes the advances in the techniques for detection of A. cantonensis in snails.

Clinical Characteristics and Treatment Outcome of Eosinophilic Meningoencephalitis in South Indian Children: Experience From a Prospective Registry.

BACKGROUND: To describe the clinical features and therapeutic outcomes of a prospective cohort of children with eosinophilic meningoencephalitis. METHODS: Children admitted with clinical features suggestive of meningitis along with cerebrospinal fluid (CSF) eosinophilia during the period of 14 years (2008 to 2021) were included. Their baseline characteristics, epidemiologic associations, and treatment outcomes were analyzed and compared with the previous studies. RESULTS: We identified 25 children (13 males) satisfying the inclusion criteria. The median age at presentation was 3.9 years (range 0.8 to 17 years); 68% were aged less than two years. Fourteen (56%) children had a history of exposure to snails. Most of them presented with fever, headache, irritability, lateral rectus palsy, and early papilledema. Symptoms started three to 42 days (median duration: 14 days) before admission to our center. All children had peripheral eosinophilia, which ranged from 9% to 41%. The mean CSF white blood cell count was 416/mm3 (range 50 to 1245 cells/mm3) with CSF eosinophilia ranging from 11% to 80%. Brain magnetic resonance imaging was done in 24 children and was normal in 15 (62.5%). Leptomeningeal enhancement was seen in two (8.3%) children, and other nonspecific changes were noted in seven (29.1%) children. All children recovered without any neurological deficits with a standard treatment regimen of albendazole and oral steroids. All were asymptomatic at the last follow-up. None of them had any recurrence during the follow-up period. CONCLUSION: We report one of the largest clinical series of children with eosinophilic meningoencephalitis from an endemic area of South India.

Immunodiagnostic Detection of Angiostrongylus cantonensis Exposure on Hawaii Island Using Isogeographic 31-kDa Antigen.

Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis worldwide, and east Hawaii Island is a hotspot for the disease in the United States. A combination of glycoproteins with molecular weight of 31 kDa has been used as antigen to evaluate antibody response in human serum samples in Thailand with high specificity and sensitivity. In a previous pilot study, the Thailand-isolated 31-kDa proteins showed efficacy in dot-blot tests using serum samples from 435 human volunteers on Hawaii Island. However, we hypothesized that native antigen isolated from Hawaii A. cantonensis may exhibit higher specificity than the Thailand-isolated 31-kDa antigen due to potential minor variation in epitopes between isolates. In this study, 31-kDa glycoproteins were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis from adult A. cantonensis nematodes collected from rats captured on east Hawaii Island. The resultant proteins were purified by electroelution, pooled, bioanalyzed, and quantified. A subset of 148 samples from human participants of the original cohort of 435 was consented for this study, including 12 of the original 15 clinically diagnosed participants. Results of ELISA using the Hawaii-isolated 31-kDa antigen were compared with results of the same serum samples previously tested with both crude Hawaii antigen ELISA and Thailand 31-kDa antigen dot blot. This study shows a seroprevalence in the general population of East Hawaii Island of 25.0%, similar to previous findings of 23.8% seroprevalence in this cohort using crude antigen from Hawaii A. cantonensis and 26.5% using Thailand 31-kDa antigen.

Clinical Characteristics of Long-Term Complications of Severe Rat Lung Worm Disease in Hawai'i: A Survey of 4 Cases.

Rat lung worm disease (RLWD) is endemic to Hawai'i, and cases of severe RLWD with long-term sequelae have been reported in Hawai'i. However, there are limited data on clinical features of the RLWD survivors with the long-term sequelae. The authors conducted a survey to report on clinical characteristics of RLWD survivors with the long-term sequelae. Four RLWD survivors had severe RLWD with persistent, neurological symptoms for years after RLWD. In conclusion, long-term sequelae of severe RLWD exist. The most common long-term consequence among participants was severe skin pain, which may relate to damage of the nerves or spinal cord.

High-throughput metagenomics analysis in early and precise diagnosis of eosinophilic meningoencephalitis complicated with respiratory failure: A case report.

RATIONALE: Human infection with Angiostrongylus cantonensis is uncommon and has only been sporadically reported in the literature. Patients infected with A cantonensis usually have a delayed diagnosis and sometimes a poor prognosis. PATIENT CONCERNS: A 70-year-old woman presented to the respiratory department with complaints of headache, chest pain, myalgia, fatigue, and anorexia for 7 days. DIAGNOSES: Complete blood count showed eosinophilia. The serum was tested showing a positive finding of A cantonensis antibody. Cerebrospinal fluid was tested using high-throughput metagenomics analysis and 16 reads for A cantonensis were mapped. The patient was diagnosed with A cantonensis infection. INTERVENTIONS: The patient received a 7-day course of albendazole and 4-day course of prednisone. OUTCOMES: When discharged from the hospital, the patient still suffered from fatigue and poor memory. Aminotransferase levels were high due to albendazole's liver toxicity. In a post-discharge follow-up about 1 month later she had recovered completely both physically and mentally, and peripheral eosinophil count and aminotransferase levels were both normal. LESSONS: Because the direct identification of parasites is difficult, high-throughput metagenomics analysis may provide a reliable alternative tool for the diagnoses of infection with A cantonensis. When albendazole is prescribed, caution must be taken with respect to its liver toxicity.

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid.

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. CONCLUSIONS/SIGNIFICANCE: The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.

Application of the Midi Parasep® SF technique for the detection of L1 larvae of Angiostrongylus cantonensis in faeces of infected rats.

We investigated parasitic zoonoses caused by protozoans and helminths in urban and peri-urban rat populations (Rattus norvegicus and R. rattus) in Spanish cities. Rats were trapped and then dissected to remove adult helminths, and the contents of the large intestine were retrieved for the study of parasitic forms. The Midi Parasep® solvent free (SF) technique was used to concentrate the parasites in the intestinal contents. Some of the rats studied (n = 8) were infected by the rat lungworm, Angiostongylus cantonensis, whose first stage larvae (L1) are shed in rat faeces. After the concentration technique, L1 larvae were found in the sediment of 6 of the 8 positive rats. The two negative sediment samples were due to the presence of either only adult females or, in addition to males, only young females in the lungs of the rats. In view of our results, Midi Parasep® SF turned out to be a simple, rapid, inexpensive, and sensitive method to detect nematode larvae, such as the L1 larvae of A. cantonensis (or A. costaricensis), in natural and experimentally infected rats.

The therapeutic effect of tanshinone IIA in mouse astrocytes after treatment with Angiostrongylus cantonensis fifth-stage larval excretory-secretory products.

BACKGROUND: Angiostrongylus cantonensis is an important food-borne zoonotic parasite that causes eosinophilic meningitis and meningoencephalitis in humans. Excretory-secretory products (ESPs) are valuable targets for studying host-parasite relationships. ESPs are composed of a variety of molecules that are used to penetrate defensive barriers and avoid immune attack of the host. Tanshinone IIA (TSIIA) is a vasoactive cardioprotective drug that is widely used in studies evaluating potential therapeutic mechanisms. In this study, we will evaluate the therapeutic effects of TSIIA in mouse astrocytes after A. cantonensis fifth-stage larvae (L5) ESPs treatment. METHODS: Here, we examined the therapeutic effect of TSIIA by real-time qPCR, western blotting, activity assay, and cell viability assays. RESULTS: First, the results showed that TSIIA can elevate cell viability in astrocytes after stimulation with ESPs. On the other hand, TSIIA downregulated the expression of apoptosis-related molecules. However, the expression of molecules related to antioxidant, autophagy, and endoplasmic reticulum stress was significantly increased. The results of antioxidant activation assays showed that the activities of superoxide dismutase (SOD), glutathione S-transferase (GST), and catalase were significantly increased. Finally, we found that cell apoptosis and oxidative stress were reduced in TSIIA-treated astrocytes by immunofluorescence staining. CONCLUSION: The findings from this study suggest that TSIIA can reduce cellular damage caused by A. cantonensis L5 ESPs in astrocytes and clarify the related molecular mechanisms.

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.

Improving the detection of Angiostrongylus cantonensis in the brain tissues of mammalian hosts.

Angiostrongylus cantonensis is an invasive nematode parasite that causes eosinophilic meningitis in many vertebrate hosts, including humans. This parasite is spreading rapidly through the six continents, with Europe being the final frontier. Sentinel surveillance may be a cost-effective strategy to monitor the pathogen's arrival to new geographical regions. Necropsy, followed by tissue digestion, is often used to recover helminth parasites from vertebrate host tissues, however, to detect brain parasites, this protocol is poorly utilized. Our brain digestion protocol is easily performed and 1) reduces false positivity and negativity, 2) provides accurate estimates of parasite burden and 3) helps establish a more precise prevalence. Early detection of A. cantonensis increases the efficacy of prevention, treatment, and disease control strategies for susceptible animal populations and humans.

CEBPα/miR-101b-3p promotes meningoencephalitis in mice infected with Angiostrongylus cantonensis by promoting microglial pyroptosis.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) infection can induce acute inflammation, which causes meningoencephalitis and tissue mechanical injury to the brain. Parasite infection-induced microRNAs play important roles in anti-parasite immunity in non-permissive hosts. miR-101b-3p is highly expressed after A. cantonensis infection; however, the role of miR-101b-3p and the transcription regulation of miR-101b-3p in A. cantonensis infection remain poorly characterized. RESULTS: In the present study, we found that miR-101b-3p inhibition alleviated inflammation infiltration and pyroptosis in A. cantonensis infection. In addition, we found that CCAAT/enhancer-binding protein alpha (CEBPα) directly bound to the - 6-k to - 3.5-k region upstream of miR-101b, and CEBPα activated miR-101b-3p expression in microglia. These data suggest the existence of a novel CEBPα/miR-101b-3p/pyroptosis pathway in A. cantonensis infection. Further investigation verified that CEBPα promotes pyroptosis by activating miR-101b-3p expression in microglia, and microglial pyroptosis further promoted inflammation. CONCLUSIONS: Our results suggest that a CEBPα/miR-101b-3p/pyroptosis pathway may contribute to A. cantonensis infection-induced inflammation and highlight the pro-inflammatory effect of miR-101b-3p. Video Abstract.

The invasive giant African land snail, Achatina fulica (Gastropoda: Pulmonata): global geographical distribution of this species as host of nematodes of medical and veterinary importance.

The giant African land snail, Achatina fulica, is an important invasive species in many countries, where it causes losses in biodiversity and agriculture, as well as impacting the health of both humans and animals, as the intermediate host of medically important nematodes. The present study is based on a comprehensive review of the literature on the nematodes that have been found in association with A. fulica, worldwide. We searched a number of different databases and used the findings to investigate the methods used to extract and identify the nematodes, their larval stages, and environment and collecting procedures of the infected molluscs. Between 1965 and 2021, 11 nematode species were recorded in association with A. fulica in 21 countries. Most of the studies recorded associations between A. fulica and Angiostrongylus cantonensis, which causes cerebral angiostrongyliasis in humans and Aelurostrongylus abstrusus, which provokes pneumonia in felines. The nematodes were extracted primarily by artificial digestion with hydrochloric acid or pepsin, and identified based on their morphology or through experimental infection to obtain the adult. In most cases, the nematodes were at larval stage L3, and the infected A. fulica were collected from anthropogenic environments. The results demonstrate the importance of A. fulica as a host of nematodes of medical and veterinary importance, as well the contribution of anthropogenic environments to the occurrence of the parasites, and give information about the different methods used to collect and identify the nematodes found associated with this species.

The application of metagenomic next-generation sequencing for Angiostrongylus eosinophilic meningitis in a pediatric patient: A case report.

Background: Angiostrongylus eosinophilic meningitis (AEM) is a rare yet emerging disease caused by Angiostrongylus cantonensis infection. Its atypical symptoms may delay the diagnosis and cause fatal outcomes, especially in the early stages of infection and among children. Case presentation: Here we reported the use of metagenomic next-generation sequencing (mNGS) to facilitate the diagnosis and treatment of an 8-year-old boy with severe A. cantonensis infection. The mNGS tests consistently identified the infection of A. cantonensis prior to the detection by the immunologic method and confirmed it as AEM. Owing to the multidisciplinary team (MDT)-administrated treatments and close disease monitoring based on regular clinical tests and sequential mNGS tests, the patients eventually fully recovered from severe infectious conditions. Conclusion: This case demonstrated the advantages of mNGS for early diagnosis of AEM in pediatric patients, highlighting its application for pan-pathogen detection, as well as disease monitoring for severe A. cantonensis infection.

Autochthonous Angiostrongylus cantonensis Lungworms in Urban Rats, Valencia, Spain, 2021.

To determine the role of rats as potential reservoirs of zoonotic parasites, we examined rats trapped in urban sewers of Valencia, Spain, in 2021. Morphologic and molecular identification and sequencing identified autochthonous Angiostrongylus cantonensis nematodes, the most common cause of human eosinophilic meningitis, in pulmonary arteries of Rattus norvegicus and R. rattus rats.

Immunoanalysis of different antigenic preparations of Angiostrongylus cantonensis for neuroangiostrongyliasis diagnosis improvement.

BACKGROUND: Angiostrongylus cantonensis is the etiological agent of neuroangiostrongyliasis in humans, which is developed in gastropods and vertebrate species, mainly rodents. Human transmission occurs through consumption of molluscs and paratenic hosts infected with L3, and the migration of larvae to the central nervous system causes eosinophilic meningitis. Laboratory diagnosis is based on molecular and immunological tests, using young or adult females as a source of antigens. However, these tests give positive results only after several weeks of symptoms onset and also cross-reactions with others parasite infections may occur. OBJECTIVES: The purpose of this work was to study different antigenic preparations of distinct evolutionary phases of A. cantonensis, in order to improve serological techniques for disease immunodiagnosis. METHODS: For this purpose, antigenic fractions of different evolutionary forms were evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) and Western blot using serum samples. FINDINGS: All analysed fractions showed reactivity with serum samples from patients with neuroangiostrongyliasis, especially female membrane alkaline (FAM) and female soluble alkaline (FAS) fractions together with female soluble saline (FSS), improving the technique specificity. MAIN CONCLUSIONS: The results point to the possibility of use of raw female antigens in association with alkaline membrane antigens extracted from adult worms to aid in diagnosis and helps initiate neuroangiostrongyliasis surveillance and control actions.

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.