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.

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.

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.

IL-17A neutralizing antibody attenuates eosinophilic meningitis caused by Angiostrongylus cantonensis by involving IL-17RA/Traf6/NF-κB signaling.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) is a foodborne parasite that can invade the central nervous system (CNS), resulting in eosinophilic meningitis (EM). However, the mechanism by which A. cantonensis causes eosinophilic infiltration into CNS is not well understood. METHODS: In this study eosinophilic infiltration into the CNS caused by A. cantonensis was assessed based on eosinophil counts and evaluation of interleukin (IL)-5 and -13 levels by real-time PCR in brain of Balb/c mice. The expression and activation of IL-17A, IL17 receptor (IL-17R A), and IL-17RC and the related signaling molecules nuclear factor (NF)-κB1, NF-κB2, NF-κB activator (Act)1, tumor necrosis factor receptor-associated factor (Traf)5, and Traf6 during A. cantonensis infection in brain tissue of Balb/c mice were examined by real-time, western blotting and immunofluroence. A. cantonensis-infected Balb/c mice were treated with IL-17A neutralizing antibody to evaluate the role of IL17A in eosinophil accumulation in the CNS. RESULTS: Our results showed A. cantonensis infection caused eosinophil accumulation and alterations in IL-5 and -13 levels. The expression of IL-17A and -17RA, Act1, and Traf6 but not of IL-17RC and Traf5 was upregulated during infection; this was accompanied by NF-κB1 and -κB2 activation. Importantly, application of IL-17A neutralizing antibody attenuated eosinophil accumulation in CNS and reversed the changes in IL-5 and -13 expression caused by A. cantonensis infection. Additionally, IL-17RA and Traf6 levels decreased, which was accompanied by NF-κB inactivation. CONCLUSION: IL-17A plays an important role in EM caused by A. cantonensis, possibly through activation of NF-κB via the IL-17RA/Traf6 signaling pathway. These findings highlight the potential for using IL-17A neutralizing antibody as a therapeutic strategy for the treatment of EM.

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.

Dexamethasone Downregulates Expressions of 14-3-3ß and γ-Isoforms in Mice with Eosinophilic Meningitis Caused by Angiostrongylus cantonensis Infection.

Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infections. The mechanism steroids act on eosinophilic meningitis remains unclear. In this mouse experiments, expressions of 14-3-3 isoform ß and γ proteins significantly increased in the CSF 2-3 weeks after the infection, but not increasedin the dexamethasone-treated group. Expression of 14-3-3 ß, γ, ε, and θ isoforms increased in brain meninges over the 3-week period after infection and decreased due to dexamethasone treatment. In conclusion, administration of dexamethasone in mice with eosinophilic meningitis decreased expressions of 14-3-3 isoform proteins in the CSF and in brain meninges.

Angiostrongylus cantonensis Infection of Central Nervous System, Guiana Shield.

We report a case of eosinophilic meningitis complicated by transverse myelitis caused by Angiostrongylus cantonensis in a 10-year-old boy from Brazil who had traveled to Suriname. We confirmed diagnosis by serology and real-time PCR in the cerebrospinal fluid. The medical community should be aware of angiostrongyliasis in the Guiana Shield.

The mouse cortical meninges are the site of immune responses to many different pathogens, and are accessible to intravital imaging.

A wide range of viral and microbial infections are known to cause meningitis, and there is evidence that the meninges are the gateway to pathogenic invasion of the brain parenchyma. Hence observation of these regions has wide application to understanding host-pathogen interactions. Interactions between pathogens and cells of the immune response can be modified by changes in their environment, such as suppression of the flow of blood and lymph, and, particularly in the case of the meninges, with their unsupported membranes, invasive dissection can alter the tissue architecture. For these reasons, intravital imaging through the unperforated skull is the method of choice. We give a protocol for a simple method of two-photon microscopy through the thinned cortical skull of the anesthetized mouse to enable real-time imaging with sub-micron resolution through the meninges and into the superficial brain parenchyma. In reporter mice in which selected cell types express fluorescent proteins, imaging after infection with fluorescent pathogens (lymphocytic choriomeningitis virus, Trypanosoma brucei or Plasmodium berghei) has shown strong recruitment to the cortical meninges of immune cells, including neutrophils, T cells, and putative dendritic cells and macrophages. Without special labeling, the boundaries between the dura mater, the leptomeninx, and the parenchyma are not directly visualized in intravital two-photon microscopy, but other landmarks and characteristics, which we illustrate, allow the researcher to identify the compartment being imaged. While most infectious meningitides are localized mainly in the dura mater, others involve recruitment of immune cells to the leptomeninx.

Eosinophilic Meningitis Due to Infection With Paragonimus kellicotti.

Paragonimus kellicotti is an emerging pathogen in the United States with 19 previously reported cases, most in Missouri. Pulmonary symptoms with eosinophilia are most common, though 1 case did involve the central nervous system with few symptoms. We describe the first 2 cases of eosinophilic meningitis due to Paragonimus kellicotti.

Angiostrongylus cantonensis Is an Important Cause of Eosinophilic Meningitis in Southern Vietnam.

We utilized polymerase chain reaction (PCR) to demonstrate that Angiostrongylus cantonensis was responsible for 67.3% of 55 cases of eosinophilic meningitis from a cohort of 1,690 adult patients with CNS infection at a tertiary hospital in southern Vietnam. Longer duration of illness, depressed consciousness, and peripheral blood eosinophilia were associated with PCR positivity.

Angiostrongylus cantonensis DNA in Cerebrospinal Fluid of Persons with Eosinophilic Meningitis, Laos.

Definitive identification of Angiostrongylus cantonensis parasites from clinical specimens is difficult. As a result, regional epidemiology and burden are poorly characterized. To ascertain presence of this parasite in patients in Laos with eosinophilic meningitis, we performed quantitative PCRs on 36 cerebrospinal fluid samples; 4 positive samples confirmed the parasite's presence.

Autochthonous Case of Eosinophilic Meningitis Caused by Angiostrongylus cantonensis, France, 2016.

We report a case of a 54-year-old Moroccan woman living in France diagnosed with eosinophilic meningitis caused by Angiostrongylus cantonensis. Diagnosis was based on clinical symptoms and confirmed by testing of serum and cerebrospinal fluid samples. Physicians should consider the risk for A. cantonensis infection outside of endemic areas.

Angiostrongylus cantonensis Meningitis and Myelitis, Texas, USA.

Infection with Angiostrongylus cantonensis roundworms is endemic in Southeast Asia and the Pacific Basin. A. cantonensis meningitis and myelitis occurred in summer 2013 in a child with no history of travel outside of Texas, USA. Angiostrongyliasis is an emerging neurotropic helminthic disease in Texas and warrants increased awareness among healthcare providers.

Study on the tolerance and adaptation of rats to Angiostrongylus cantonensis infection.

Angiostrongylus cantonensis (A. cantonensis) is the most common infectious agent causing eosinophilic meningitis. As an important food-borne parasitic disease, angiostrongyliasis cantonensis is an emerging infectious disease which brings severe harm to central nerve system of human. Rat, one of the few permissive hosts of A. cantonensis known to date, plays an indispensable role in the worm's life cycle. However, the tolerance and adaptation of rat to A. cantonensis infection is rarely understood. In this study, we infected rats with different numbers the third stage larvae (L3) of A. cantonensis and explored their tolerance through analysis on survival curve, neurological function score, and detection of pathological damages in organs including the brain, lung, and heart of the animals. Results indicated that rats' survival condition worsens, and body weight dropped more significantly as more worms were used for infection. Death appeared in groups infected with 80 and more A. cantonesnsis per rat. Morris water maze revealed that the neurological function of rats damaged gradually with increasing infection number of A. cantonensis larvae. When the number of infected parasite exceeded 240 per animal, rats showed significant neurological impairments. Collection of A. cantonensis from rat lung after 35 days of infection implied an upper limit for worm entry, and the average length of worm was inversely proportional to the infection amount, while the ratio between female and male worms was positively related to the infection number. The degree of pulmonary and cardiac inflammation was proportional to the infection number of A. cantonensis. Meanwhile, there existed considerable amount of adult worms in rat's right atrium and right ventricle, leading to a right heart myocardial inflammation. The present study firstly reports the tolerance and adaptation of rat, a permissive host of A. cantonensis to its infection, which will not only provide accurate technical parameters for maintaining A. cantonensis life cycle under laboratory conditions but also help unveil the underlying mechanism of the distinct pathological outcomes in the permissive and non-permissive hosts with A. cantonensis infection.

Angiostrongylus cantonensis Infection: A Cause of Fever of Unknown Origin in Pediatric Patients.

Fever of unknown origin (FUO) in children is frequently caused by infectious diseases. Angiostrongylus cantonensis, while a primary cause of eosinophilic meningitis, is rarely a cause of FUO. We present 2 pediatric cases of FUO caused by Angiostrongylus cantonensis acquired in Houston, Texas, outside its usual geographic distribution.

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.

Temporal-spatial pathological changes in the brains of permissive and non-permissive hosts experimentally infected with Angiostrongylus cantonensis.

Human cerebral angiostrongyliasis becomes an emerging disease in many parts of the world. By postmortem examination, Angiostrongylus cantonensis have been reported to cause severe pathological changes in the central nervous system. The present study was designed to determine the temporal-spatial pathological changes through experimental infections and histopathological examination of permissive (SD rats) and non-permissive (ICR mice) hosts. After infecting SD rats with 25, 50, or 100 third-stage larvae (L3) and ICR mice with 25 L3, one animal from each group was sacrificed daily from day 1 to day 30 post-infection. Each rat brain was cut into six sections and mouse brain into five sections. These sections were stained with haematoxylin and eosin and examined microscopically. Eosinophilic meningitis was found to be the most commonly pathological change and occurred on day 17 post-infection in rats with 25 L3, day 9 in the 50- or 100-L3 groups, and day 12 in infected mice. Thickness of the meninges increased 9-24 folds in infected rats and 89 folds in an infected mouse on day 28. Encephalitis, congestion, perivascular cuffing, and haemorrhage were revealed in infected mice and rats with 100 L3. Fifth-stage larvae were frequently observed in the meninges but occasionally in the parenchyma. Significant correlations between meningitis and presence of larvae in the meninges were found in the three infected rat groups but not in the infected mice. The results indicate that the clinical course of A. cantonensis infection is not self-limited but becomes more severe with the intensity of infection.

Fibronectin changes in eosinophilic meningitis with blood-CSF barrier disruption.

Fibronectin, which is present at relatively low levels in healthy central nervous systems (CNS), shows increased levels in meningitis. In this study, fibronectin processing was correlated with the increased permeability of the blood-cerebrospinal fluid (CSF) barrier as well as with the formation of eosinophil infiltrates in angiostrongyliasis meningitis. The immunohistochemistry results show matrix metalloproteinase-9 (MMP-9) is localized in the choroid plexus epithelium. Coimmunoprecipitation demonstrated fibronectin strongly binds MMP-9. Furthermore, treatment with the MMP-9 inhibitor GM6001 significantly inhibited fibronectin processing, reduced the blood-CSF barrier permeability, and decreased the eosinophil counts. The decreased fibronectin processing in CSF implies decreased cellular invasion of the subarachnoid space across the blood-CSF barrier. Therefore, increased fibronectin processing may be associated with barrier disruption and participate in the extravasation and migration of eosinophils into the CNS during experimental parasitic infection.