An indirect immunofluorescence antibody test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis

Abdominal angiostrongyliasis is a potentially fatal zoonotic disease with a broad geographical distribution throughout Central and South America. This study assessed the performance of Angiostrongylus costaricensis eggs as the antigen in an indirect immunofluorescence assay for the determination of parasite-specific IgG and IgG1 antibodies. For prevalence studies, an IgG antibody titre > 16 was identified as the diagnostic threshold with the best performance, providing 93.7 percent sensitivity and 84.6 percent specificity. Cross reactivity was evaluated with 65 additional samples from patients with other known parasitic infections. Cross reactivity was observed only in samples from individuals infected with Strongyloides stercoralis. For clinical diagnosis, we recommend the determination of IgG only as a screening test. IgG1 determination may be used to increase the specificity of the results for patients with a positive screening test.

Use of heterologous antigens for the immunodiagnosis of abdominal angiostrongyliasis by an enzyme-linked immunosorbent assay

Angiostrongylus costaricensis has a broad geographic distribution spanning from North to South America and the infections of vertebrates with this nematode can result in abdominal complications. Human infections are diagnosed by histological or serological methods because the isolation of larvae from feces is not feasible, as most parasites become trapped in intestinal tissues due to intense eosinophilic inflammation. Because A. costaricensis is difficult to maintain in the laboratory, an immunodiagnostic IgG enzyme-linked immunosorbent assay (ELISA) using antigens from the congeneric Angiostrongylus cantonensis species was evaluated against a panel of serum samples from patients who were histologically diagnosed with A. costaricensis infections. Sera from uninfected individuals and individuals infected with other parasites were used as controls. The sensitivity and specificity of the assay were estimated at 88.4 percent and 78.7 percent, respectively. Because the use of purified or cloned antigens has not been established as a reliable diagnostic tool, the use of heterologous antigens may provide a viable alternative for the development of an ELISA-based immunodetection system for the diagnosis of abdominal angiostrongyliasis.

Eosinophilic meningitis associated with angiostrongyliasis: clinical features, laboratory investigations and specific diagnostic IgG and IgG subclass antibodies in cerebrospinal fluid.

Eosinophilic meningitis (EOM) associated angiostrongyliasis mostly induced by the nematode Angiostrongylus cantonensis, is a common disease with worldwide prevalence. Heavy infections can lead to chronic disabling disease and even death. This study was conducted to shed light on the overall specific IgG antibody response as well as the specific IgG antibody subclass responses in cerebrospinal fluid (CSF) of patients with EOM. Fifteen patients with EOM associated with angiostrongyliasis were included in the study. Sera were screened by immunoblotting for the presence of IgG antibody to the 29 kDaA. cantonensis antigenic polypeptide. CSF was examined by ELISA for the presence of specific IgG and IgG subclass antibodies. Patients presented with headache (100%), neck stiffness (20%), fever (40%), nausea (87%), vomiting (73%), paresthesia (7%), and muscle weakness (7%). Seven of 15 (47%) patients showed peripheral blood eosinophilia and all patients presented with eosinophils in CSF. A sensitivity of 80 % was obtained by combining the diagnostic values of immunoblotting in sera and IgG and IgG subclasses-based ELISA in CSF. The combination of a history of eating raw or semi-cooked infected foods, clinical features, complete blood count, differential cell counts, CSF profiles, and serum and CSF antibodies to A. cantonensis can be used to increase the sensitivity for the diagnosis of human angiostrongyliasis.

Angiostrongylus costaricensis, A. cantonensis e A. vasorum: diferenciação, detecção no hospedeiro intermediário e filogenia utilizando a PCR-RFLP, Multiplex PCR e sequenciamento da região espaçadora transcrita interna do DNA ribossomal / Angiostrongylus costaricensis, A. cantonensis and A. vasorum: differentiation, tracing the intermediate host and phylogeny using PCR-RFLP, multiplex PCR and sequencing of internal transcribed spacer of ribosomal DNA

Angiostrongylus costaricensis, A. cantonensis e A. vasorum possuem importância humana e veterinária. O exame dos moluscos infectados com esses parasitos é feito após digestão do molusco e sedimentação das larvas (L3). Essas larvas são inoculadas em roedores para obtenção dos vermes adultos que são identificados morfologicamente. Em decorrência dessas dificuldades, desenvolvemos um método molecular para a detecção de larvas desses parasitos utilizando a técnica de PCR-RFLP com iniciadores direcionados para as regiões conservadas da região espaçadora transcrita interna dois do DNA ribossomal (ITS2 do rDNA nuclear) e citocromo oxidase subunidade I do DNA mitocondrial (COI do DNAmt). A enzima de restrição ClaI foi a que forneceu os resultados mais precisos para a primeira região e a RsaI para a segunda. Para a reconstrução filogenética desses helmintos sequenciamos a região ITS2 do rDNA. As topologias das árvores foram ligeiramente diferentes. Na árvore de máxima parcimônia (MP) foi observada a formação de dois grupos: I) A. costaricensis, II) A. cantonensis e A. vasorum. Nas árvores de agrupamento de vizinhos (NJ) e máxima verossimilhança (ML) também houve a formação de dois grupos: I) A. costaricensis e A. vasorum, II) A. cantonensis. Nas três árvores, o grupo de A. costaricensis foi dividido em dois ramos, separando as populações do Brasil e da Costa Rica, sustentados por alto valor de “bootstrap”. As topologias das árvores e os valores de distância dessas populações sugerem que elas estão se diferenciando, provavelmente, devido à distância geográfica. Os resultados obtidos sugerem a existência de um único gênero: Angiostrongylus. De fato, as espécies A. cantonensis e A. costaricensis consideradas por alguns autores como pertencentes ao gênero Parastrongylus mostraram-se distantes entre si e mais próximas de A. vasorum. A partir das sequências desses nematóides, foram desenhados dois iniciadores. Esses iniciadores associados a um par de iniciador direcionado para a região ITS do hospedeiro intermediário, foram utilizados em uma única reação, denominada multiplex-PCR. Foram obtidos perfis específicos para cada nematóide. O amplicon de ITS2 de A. costaricensis do Brasil foi de 400 pb, de A. costaricensis da Costa Rica 480 pb, de A. cantonensis um fragmento duplo de 480-500 pb e de A. vasorum 450 pb. Com essa mesma metodologia detectou-se A. costaricensis em Sarasinula marginata experimentalmente infectada, sendo obtido um amplicon do parasito de 400 pb, associado ao amplicon do molusco de 1000 pb.

Angiostrongylus costaricensis, A. cantonensis e A. vasorum: Diferenciação, detecção no hospedeiro intermediário e filogenia utilizando a PCR-RFLP, Multiplex PCR e sequenciamento da região espaçadora transcrita interna do DNA ribossomal

Angiostrongylus costaricensis, A. cantonensis e A. vasorum possuem importância humana e veterinária. O exame dos moluscos infectados com esses parasitos é feito após digestão do molusco e sedimentação das larvas (L3). Essas larvas são inoculadas em roedores para obtenção dos vermes adultos que são identificados morfologicamente. Em decorrência dessas dificuldades, desenvolvemos um método molecular para a detecção de larvas desses parasitos utilizando a técnica de PCR-RFLP com iniciadores direcionados para as regiões conservadas da região espaçadora transcrita interna dois do DNA ribossomal (ITS2 do rDNA nuclear) e citocromo oxidase subunidade I do DNA mitocondrial (COI do DNAmt). A enzima de restrição ClaI foi a que forneceu os resultados mais precisos para a primeira região e a RsaI para a segunda. Para a reconstrução filogenética desses helmintos sequenciamos a região ITS2 do rDNA. As topologias das árvores foram ligeiramente diferentes. Na árvore de máxima parcimônia (MP) foi observada a formação de dois grupos: I) A. costaricensis, II) A. cantonensis e A. vasorum. Nas árvores de agrupamento de vizinhos (NJ) e máxima verossimilhança (ML) também houve a formação de dois grupos: I) A. costaricensis e A. vasorum, II) A. cantonensis

Nas três árvores, o grupo de A. costaricensis foi dividido em dois ramos, separando as populações do Brasil e da Costa Rica, sustentados por alto valor de “bootstrap”. As topologias das árvores e os valores de distância dessas populações sugerem que elas estão se diferenciando, provavelmente, devido à distância geográfica. Os resultados obtidos sugerem a existência de um único gênero: Angiostrongylus. De fato, as espécies A. cantonensis e A. costaricensis consideradas por alguns autores como pertencentes ao gênero Parastrongylus mostraram-se distantes entre si e mais próximas de A. vasorum. A partir das sequências desses nematóides, foram desenhados dois iniciadores. Esses iniciadores associados a um par de iniciador direcionado para a região ITS do hospedeiro intermediário, foram utilizados em uma única reação, denominada multiplex-PCR. Foram obtidos perfis específicos para cada nematóide. O amplicon de ITS2 de A. costaricensis do Brasil foi de 400 pb, de A. costaricensis da Costa Rica 480 pb, de A. cantonensis um fragmento duplo de 480-500 pb e de A. vasorum 450 pb. Com essa mesma metodologia detectou-se A. costaricensis em Sarasinula marginata experimentalmente infectada, sendo obtido um amplicon do parasito de 400 pb, associado ao amplicon do molusco de 1000 pb

Detection of Angiostrongylus malaysiensis circulating antigen using monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA).

Three MAbs 1C4.2D8, 1C4.2C4 and 1C4.1F5 were produced using sonicated adult worm antigens of Angiostrongylus malaysiensis and they were found to be secreters of IgG1. The MAbs 1C4.2C4 and 1C4.2D8 were found to react with antigens of A. malaysiensis and cross-react with the closely related A. cantonensis but not with other helminths. A total of 108 human sera collected from Orang Asli (aborigenes) from Grik, in the State of Perak were tested for A. malaysiensis infection using the MAb-ELISA. MAb 1C4.1F5 and 25 (23%) were positive. Twenty of these positive samples were tested with the MAb 1C4.2D8 and none was found to be positive.

Specificity of antibodies in cerebrospinal fluid of human cerebral gnathostomiasis cases.

Specificity of antibodies in cerebrospinal fluid (CSF) of human cerebral gnathostomiasis cases were examined by indirect fluorescent antibody technique against paraffin sections of Gnathostoma spinigerum larva. Specific greenish fluorescence was observed at cuticle, esophagus, muscle cells, intestinal cell cytoplasm and microvilli. CSF of confirmed cerebral cysticercosis cases gave fluorescence mostly at the cuticle. It is suggested that parasite-specific antigen may be present on intestinal cell microvilli and CSF would be a good source of antibodies in studying specificity of antibodies to gnathostome infections.

Humoral immune response of Balb/c mice to larval and adult worm antigens of Angiostrongylus malaysiensis.

The development of Angiostrongylus malaysiensis in Balb/c mice and the humoral response due to it were studied by using the enzyme-linked immunosorbent assay (ELISA) with adult worm and L3 antigens. The worms recovered from mice were seen in the brain tissue only, they failed to migrate to the lung as in the normal host (rats). The antibody titres of sera from infected mice, showed similar patterns in response to L3 antigen and to adult worm antigen. However, the highest antibody response could be detected by L3 antigen in the early period after infection while the adult worm antigen detected a higher response in the later stages of development.

Preparation of monoclonal antibody against Angiostrongylus cantonensis antigen.

Monoclonal antibodies (MAb) against A cantonensis were produced through fusion of immunised spleen cells from BALB/c mice with NS-1 myeloma cells at a ratio of 10:1. The successful fusion rate on the 3rd day of fusion was 90.1%. Ten MAb were characterised, six of which were IgG1 and the remaining four were IgG2a, IgG2b, IgM and IgA respectively. Among 6 IgG1 MAb, four were A. cantonensis-specific, of which three reacted to adult worm antigen only and one reacted to both adult worm and juvenile worm antigens. Two other IgG1 MAb showed cross-reaction with other helminthic antigens of Toxocara canis. Ascaris suum. Paragonimus westermani, Dirofilaria immitis, Anisakis Spp, Gnatostoma Spinigerum and Clonorchis sinensis.

Obtención y evaluación de antígenos de excresión-secreción en hembras adultas de Angiostrongylus cantonensis / Attainment and evaluation of excretion-secretion antigens in adult females of Angiostrongylus cantonensis

Hembras adultas de Angiostrongylus cantonensis se mantuvieron in vitro para la obtención de antígenos de excreción-secreción (Ag Exc-Sec Ac). Los antígenos se evaluaron frente a inmunosueros obtenidos en conejo y sueros de ratas infestadas experimentalmente, mediante las técnicas de contrainmunoelectrofóresis e inmunodifusión doble al no mostrar reaccciones cruzadas con otros helmintos. Se obtuvo un inmunosuero de alto título y especificidad. Se detectaron títulos de anticuerpos en suero de ratas infestadas en diferentes tiempos de posinfestación con antígenos de excreción-secreción de adultos

ELISA testing in cases of clinical angiostrongyliasis in Thailand.

The sera from 116 Thais admitted to Nakhon Ratchasima hospital in northeastern Thailand with eosinophilic meningitis were tested for antibodies to Angiostrongylus cantonensis by ELISA. Ninety-six percent of the sera were considered positive with ELISA values exceptionally high for most patients. The clinical aspects of the disease are also presented. The ELISA test is considered to be of value in the diagnosis of the disease but tests for the antigen would provide a more definitive diagnosis.

ELISA for the detection of angiostrongylus cantonensis antibodies in patients with eosinophilic meningitis.

The enzyme-linked immunosorbent assay (ELISA) was used to examine sera from twelve Taiwanese children with eosinophilic meningitis suspected to be induced by infection with Angiostrongylus cantonensis. Polystyrene tubes were coated with Angiostrongylus cantonensis antigens (5 micrograms/ml protein) prepared from fourth-stage larvae recovered from the brains of experimentally infected rats. Alkaline phosphatase labelled goat antihuman IgG conjugate was used in a dilution of 1 to 500; sera were diluted 1 to 1000. Positive control sera were from patients with parasitologically confirmed infections; the negative control sera from healthy persons. The ELISA values for sera from suspected cases of angiostrongyliasis ranged from 4.5 to 23.1; the positive control sera, 12.7 to 34.4 and the negative control sera 1.3 and 2.2. The assay shows promise and with the use of more purified antigens, micromethods and automated ELISA readers it should become valuable in the presumptive diagnosis of angiostrongyliasis in endemic areas.