Neuroimmunological findings of Angiostrongylus cantonensis meningitis in ecuadorian patients / Hallazgos neuroinmunologicos en meningitis por Angiostrongylus cantonesis en pacientes ecuatorianos

Meningitis caused by Angiostrongylus cantonensis has recently been reported in patients resulting from the first outbreaks in subtropical regions of Ecuador. METHOD: Eight young adult patients from the two outbreaks were studied. IgA, IgM, IgG and albumin in cerebrospinal fluid and serum were quantified and plotted in cerebrospinal fluid/serum quotient diagrams (Reibergrams). The anamnesis on the patients included asking about any consumption of raw snails, symptoms and harm caused. RESULTS: Mean eosinophilia of 7.5 percent and 26 percent in serum and cerebrospinal fluid respectively was observed, as well as a moderate increase in total proteins. The most frequent pattern of intrathecal synthesis was observed in three classes of immunoglobulins. Intrathecal synthesis of IgM was observed in all cases two weeks after the first symptoms appeared. CONCLUSION: The intrathecal synthesis patterns of eosinophilic meningitis due to Angiostrongylus cantonensis, facilitated by cerebrospinal fluid analysis, were similar to those of previous cases from abroad.

La meningitis provocada por Angiostrongylus cantonensis ha sido reportada recientemente en pacientes procedentes de los primeros brotes reportados en regiones subtropicales de Ecuador. MÉTODO: Ocho adultos jóvenes procedentes de dos brotes fueron estudiados. Se cuantificó IgA, IgM, IgG y albúmina en suero y líquido cefalorraquídeo y fueron colocados en los gráficos de las razones líquido cefalorraquídeo/suero (reibergramas). La anamnesia incluía ingestión de caracoles crudos, los síntomas y los daños provocados. RESULTADOS: Una eosinofílica promedio de 7,5 y 26 por ciento en suero y líquido cefalorraquídeo fue observada al igual que un moderado incremento en las proteínas totales. El patrón de síntesis intratecal predominante fue de tres clases de inmunoglobulinas. La síntesis intratecal de IgM se observó en todos los casos a las dos semanas después del inicio de los síntomas. CONCLUSIÓN: El patrón de síntesis intratecal de la meningitis eosinofílica por Angiostrongylus cantonensis facilitado por el análisis del líquido cefalorraquídeo fue similar a los casos previos reportados fuera del país.

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.

Abdominal angiostrongyliasis: report of two cases with different clinical presentations / Angiostrongilíase abdominal: relato de dois casos com diferentes apresentações clínicas

Abdominal angiostrongyliasis is a sporadic infectious disease caused by the nematode Angiostrongylus costaricensis. It usually presents as acute abdomen, secondary to mesenteric ischemia, and pronounced eosinophilia. In some cases its course is insidious and transient, and the diagnosis is suspicious. The disease is confirmed by the detection of A. costaricensis elements in surgical specimen. The treatment is supportive, with avoidance of antihelminthic administration due to a possible erratic migration followed by worsening of the disease. We report two cases, both with intense eosinophilia and serum IgG-ELISA positive to A. costaricensis. The first case presented ileal perforation and was surgically treated. The second one showed hepatic nodules at ultrasound and was only symptomatically treated, evolving to an apparent protracted resolution. These two cases exemplify different clinical forms of the disease, one of them with liver involvement.

A angiostrongilíase abdominal é doença esporádica decorrente da infecção pelo nematódeo Angiostrongylus costaricensis. Costuma manifestar-se como abdome agudo secundário a isquemia mesentérica, além de marcada eosinofilia. Pode também apresentar-se de forma insidiosa e transitória, exigindo alta suspeita clínica para o diagnóstico. A doença é confirmada pela identificação de elementos do A. costaricensis em peças cirúrgicas. O tratamento é apenas de suporte, devendo-se evitar o uso de anti-helmínticos pela possibilidade de migração errática do verme com piora do quadro. Aqui foram apresentados dois casos, ambos com acentuada eosinofilia e ELISA-IgG sérico positivo para A. costaricencis. O primeiro caso cursou com perfuração ileal e foi tratado cirurgicamente. O segundo caso apresentou nódulos hepáticos ao ultrassom e foi tratado sintomaticamente, evoluindo para lenta resolução. Estes dois casos exemplificam diferentes formas de apresentação clínica da doença, uma delas com envolvimento hepático.

Individual serological follow-up of patients with suspected or confirmed abdominal angiostrongyliasis

Abdominal angiostrongyliasis (AA) is a zoonotic nematode infection caused by Angiostrongylus costaricensis, with widespread occurrence in the Americas. Although the human infection may be highly prevalent, morbidity is low in Southern Brazil. Confirmed diagnosis is based on finding parasitic structures in pathological examination of biopsies or surgical resections. Serology stands as an important diagnostic tool in the less severe courses of the infection. Our objective is to describe the follow up of humoral reactivity every 2-4 weeks up to one year, in six individuals with confirmed (C) and ten suspected (S) AA. Antibody (IgG) detection was performed by ELISA and resulted in gradually declining curves of reactivity in nine subjects (56 percent) (4C + 5S), that were consistently negative in only three of them (2C + 1S) after 221, 121 and 298 days. Three individuals (2C + 1S) presented with low persistent reacitivity, other two (1C + 1S) were serologically negative from the beginning, but also presenting a declining tendency. The study shows indications that abdominal angiostrongyliasis is usually not a persistent infection: although serological negativation may take many months, IgG reactivity is usually declining along time and serum samples pairing may add valuable information to the diagnostic workout.

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.

Abdominal angiostrongyliasis: a case with severe evolution

É relatado um caso de abdome agudo causado por angiostrongilíase abdominal. Um paciente de 42 anos, previamente hígido, apresentou queixa de nove dias de dor abdominal, constipação, disúria, febre e uma massa palpável na fossa ilíaca direita. Uma laparotomia exploradora foi realizada. Após o tratamento cirúrgico o paciente apresentou graves complicações.

Longitudinal clinical and serological survey of abdominal angiostrongyliasis in Guaporé, southern Brazil, from 1995 to 1999

Angiostrongilíase abdominal é uma zoonose causada pelo Angiostrongylus costaricensis, nematódeo que se localiza no interior de vasos mesentéricos. Nosso objetivo foi de abordar vários aspectos da história natural da parasitose, num estudo longitudinal clínico-sorológico. Um total de 179 indivíduos residentes em área rural no sul do Brasil, com transmissão ativa, foram seguidos por cinco anos. Neste período foram registradas prevalências de 28,2%, 4,2%, 10%, 20,2% e 2,8% e incidências de 0%, 5,9%, 8% e 1,5%. Tanto o sexo masculino quanto o feminino foram afetados com maiores frequências na faixa etária dos 30 aos 49 anos. Em 32 indivíduos, amostras de soro foram coletadas em todas as etapas e a reatividade de IgG detectada por ELISA foi variável e geralmente não persistindo mais do que um ano. Alguns padrões individuais foram sugestivos de re-infecção. Não houve associação com a ocorrência nem de dor abdominal nem com outras enteroparasitoses e não houve nenhum caso com diagnóstico confirmado (histopatológico) da infecção. Moluscos foram encontrados portando larvas infectantes de terceiro estadio, em algumas moradias, com uma prevalência geral de 16% e baixas cargas parasitárias. Em conclusão, a angiostrongilíase abdominal no sul do Brasil pode ser uma infecção frequente, porém com baixa morbidade e reatividade sorológica de gradual declínio.

Common antigenicities between Angiostrongylus cantonensis and various species of snails.

Studies on common antigenicities were carried out by using rabbit sera immunized with Angiostrongylus cantonensis adult worms or the third stage larvae and antigens of various species of snails and vice versa by the immunoblotting technique. The results obtained are summarized as follows: (1) Common antigenicities between A. cantonensis adult worms and snails susceptible to A. cantonensis were observed in a range of molecular weights of 14.3 to 200 kDa. In Puerto Rican pigmented Biomphalaria glabrata and Achatina fulca, which had high infection rates with A. cantonensis, we recognized 15 to 16 bands against the adult worm, especially the band with a molecular weight of 29 kDa, which had a more intense reaction. (2) Common antigenicities between A. cantonensis third stage larvae and snails susceptible to A. cantonensis, were observed in a range of molecular weights of 14.3 to 97.4 kDa, especially A. fulica and B. glabrata, where we detected many bands in molecular weight range of 18.4 to 43 kDa. Based on the common antigenicities between A. cantonensis and snails susceptible to A. cantonensis, it is possible that the common antigenicities are one of the factors defining the different susceptibilities of various species of snails to A. cantonensis, and more bands are seen with increasing infection rates with A. cantonensis. Of those bands, the protein with the molecular weight of 29 kDa may be the main common antigen between the A. cantonensis adult worm, the third stage larvae and the snails susceptible to A. cantonensis.

Caracterización de antígenos de bajo peso molecular de angiostrongylus costaricensis, reconocidos durante una infección experimental en roedores / Characterization of low molecular weight antigens of angiostrongylus costaricensis recognized during an experimental infection in mice

La respuesta inmune hacia componentes de bajo peso molecular del antígeno somático de A. costaricensis fue investigada en el modelo animal experimental. Durante la fase crónica de la infección y mediante el Western blot, son detectados por la IgG e IgG1 tres antígenos inmunodominantes: uno de 20 otro de 15 y uno de 2 kDa respectivamente. Mediante el método de digestión de Edmann se obtuvo la secuencia de aminoácidos inicial de cada componente: para la banda de 15 kDa una secuencia con un 100 por ciento de homología con la enzima glutation-S-transferasa de Brugia malayi y Onchocerca volvulus, para el componete de 2kDa una secuencia con 95 por ciento de homología con la proteína Ubiquitina, y para el compontente de 20 kDa una secuencia de trece aminoácidos, que no presenta homología con alguna proteína hasta ahora descrita. En general la reacción cruzada contra estos componentes es muy débil y en el caso particular del componente de 20 kDa no se detecta ninguna reacción cruzada. Se proponen futuros estudios con sueros humanos, para verificar la utilidad de dichos antígenos en el desarrollo de una prueba diagnóstica específica para la angiostrongiliosis abdominal.

Blinded multi-laboratory evaluation of an in-house dot-blot ELISA kit for diagnosis of human parastrongyliasis.

An in-house membrane dot-blot enzyme-linked immunosorbent assay kit, for the detection of specific antibody to a 31 kDa protein of Parastrongylus cantonensis was evaluated in a blinded multi-laboratory study. The kit was provided to technologists, who were trained in its use, from nine regional laboratories of Thailand. With an identical set of 20 coded serum samples and reference positive and negative controls, the kit was found to have a diagnostic sensitivity of 100% and a diagnostic specificity of 100% in all the laboratories. There was no obvious variation in quality among five lots of the antigen-coated nitrocellulose strips evaluated. The shelf life of the kit was > or = 6 months when the test components were stored at 4 degrees C and 18 months at -20 degrees C. The enzyme-linked immunoassay dot technique is easy to perform and does not require sophisticated electrical equipment; the result is available within 3 hours. If appropriate technical training is included, the application of this dot-blot ELISA kit in clinical laboratories throughout Thailand should be possible.

Specific IgG antibody subclasses to Angiostrongylus cantonensis in patients with angiostrongyliasis.

Total IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM specific antibodies against Angiostrongylus cantonensis somatic antigen were determined by enzyme-linked immunosorbent assay (ELISA) in sera from proven human angiostrongyliasis (PA) cases, clinically suspected angiostrongyliasis cases with eosinophilic meningitis (EM) and healthy control (HC). The specific IgA antibody in each of the patient groups was significantly higher than those of the HC group (p < 0.05). The mean ELISA value of the specific IgM in the PA group was not significantly different from that of the HC group (p > 0.05). However, the mean specific IgM ELISA value in the EM group was significantly higher than that of the HC group (p < 0.05). The levels of the specific IgG and IgG subclasses in both patient groups were significantly higher than in the healthy control (HC) group (p < 0.001). Major differences were evident in the distribution of the IgG subclass antibodies between the patient groups. The IgG1 antibody demonstrated the highest sensitivity and specificity while the IgM and IgA responses were generally poor in both patient groups. The levels of the specific IgG antibody subclasses possibly explain immune responses to the parasite.

Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worms Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis.

The antigenic components of Angiostrongylus cantonensis young adult female worm somatic extract (FSE) were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The sera tested were from patients with proven angiostrongyliasis, other parasitic diseases, and healthy adults. Both the sera and cerebrospinal fluid (CSF) were tested from patients with clinical angiostrongyliasis. The CSF from patients with other neurological diseases were also included. Using SDS-PAGE, we found that the FSE comprised more than 30 polypeptides. Immunoblot analysis revealed at least 12 or 13 antigenic bands in patients with proven or clinical angiostrongyliasis, respectively. The patterns of reactivity recognized by the serum and CSF antibodies against FSE were similar. These antigenic components had molecular masses ranging from less than 14.4 to more than 94 kDa. The prominent antigenic band of 29-kDa might serve as a reliable marker for the diagnosis of angiostrongyliasis. The sensitivity, specificity, positive and negative predictive values of immunoblot analysis in this antigenic band were 55.6%, 99.4%, 83.3% and 97.4%, respectively.

Purification of a specific immunodiagnostic Parastrongylus cantonensis antigen by electroelution from SDS-polyacrylamide gels.

A 31-kDa glycoprotein antigen was purified by electrophoresing the crude extract of Parastrongylus cantonensis adult worms in a 12% SDS-polyacrylamide gel, identifying the 31-kDa component with prestained molecular weight standards, cutting the desired gel strip, and then isolating it by electroelution. Antigen fraction of 31 kDa was re-electrophoresed, transferred to a nitrocellulose membrane and found to be reactive with only the sera from patients with parastrongyliasis. No reactive band was observed with the sera from other related parasitic infections, eg, gnathostomiasis, toxocariasis, filariasis, paragonimiasis, cysticercosis and malaria, and the normal healthy control sera. This antigen fraction isolated showed 100% sensitivity and 100% specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of 31-kDa specific antibody in the sera from patients with parastrongyliasis. The P. cantonensis antigen of 31 kDa has been obtained by this means with a high degree of purity and applied successfully in conventional ELISA for the specific immunodiagnosis of human parastrongyliasis.

Angiostrongyliasis: analysis of antigens of Angiostrongylus costaricensis adult worms versus IgG from infected patients with Angiostrongylus cantonensis.

The possibility of cross-reactivity was previously investigated by indirect ELISA with sera from Angiostrongylus cantonensis infections, normal controls and A. costaricensis antigen. 5 microg/ml of crude antigen from both sexes of each species reacted with diluted serum samples (1:800) of each of 20 cases of angiostrongyliasis and normal controls, and further with anti-human IgG conjugate at 1:1,000. The mean absorbance values were evaluated as follows; normal controls showed a value of 0.033 using A. costaricensis antigen lower than (0.085) A. costaricensis antigen. Both mean values of angiostrongyliasis cases were rather close (0.491) using A. costaricensis antigen and the other antigen (0.518). The present study continued with a crude antigen of 13 A. costaricensis females and males. Serum samples were analyzed; 27 sera of angiostrongyliasis, 30 negative controls and 193 cases of other parasitic infections (91 cases of nematodiasis; 45 cases of cestodiasis; 47 cases of trematodiasis and 10 cases of HIV) and 7 cases of other brain infections. This antigen was evaluated for ELISA with a concentration of 5 microg/ml, serum dilution 1:400 and anti-human IgG conjugate at 1:2,000. The test gave sensitivity and specificity at cut-off value 0.261; 92.59% and 73% respectively. The antigen was cross-reactive with 30 cases from 9 out of 10 different kinds of nematodiasis (gnathostomiasis, strongyloidiasis, ascariasis, hookworm infections, trichinosis, toxocariasis, trichuriasis, onchocercosis and Wuchereria bancrofti infections. Five cases from 3 of 6 kinds of cestodiasis (neurocysticercosis, echinococcosis and Hymenolepis nana infections) and 18 cases of 4 out of 5 kinds of trematodiasis (Paragonimus heterotremus infections, opisthorchiasis, schistosomiasis and fascioliasis). One case of other brain infections was observed. The crude antigen of A. costaricensis showed a high percentage sensitivity with serum antibodies of angiostrongyliasis cases. Low specificity of the test was observed by reactions of those serum antibodies with various kinds of antigenic molecules. This study provides baseline data for further immunodiagnosis of human angiostrongyliasis.

Detection of circulating antigens of Parastrongylus cantonensis in human sera by dot-blot ELISA and sandwich ELISA using monoclonal antibody.

A dot-blot ELISA was compared with a previously performed sandwich ELISA for the detection of Parastrongylus cantonensis antigens in sera from patients. Using the same monoclonal antibody and the same sera, 6 of 10 sera (60%) from parastronglyiasis patients were positive in dot-blot ELISA, whereas with sandwich ELISA, 5 of the same patient sera (50%) were positive. The specificity in both assays was 100% using 50 sera from patients with other parasitic diseases; of these, 10 each were from patients with cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 sera from normal health Thais and Malaysians. The sensitivity of the assays was, however, slightly better with dot-blot ELISA and because it is simple, quick and cost-effective, it may be a test of choice for specific diagnosis of human parastrongyliasis.

Detection of circulating antigens of Parastrongylus cantonensis in human sera by sandwich ELISA with specific monoclonal antibody.

A specific monoclonal antibody (AW-3C2) as revealed by ELISA was produced against the adult worm antigens of Parastrongylus cantonensis and used in a sandwich ELISA for the detection of circulating antigens in the sera of parastrongyliasis patients and those with other parasitic diseases. A total of 60 sera was used in this study. Of these, 10 each were from patients with parastrongyliasis filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 serum samples from normal healthy Thais and Malaysians. The mean +/- optical density (OD) values for the normal Thai and Malaysian groups were 0.126 +/- 0.028 and 0.124 +/- 0.029, respectively. Mean OD values of parastrongyliasis patient group differed significantly from that of the normal groups as well as those of other parasitic infections. Using a cut-off point of mean OD +/- 3SD of the normal control groups as indicating a positive reading, the specificity of the assay with this monoclonal antibody was 100% while the sensitivity was 50%.

The evaluation of the 29 and 31 kDa antigens in female Angiostrongylus cantonensis for serodiagnosis of human angiostrongyliasis.

The objective of this study was to evaluate the crude somatic extract of female Angiostrongylus cantonensis by ELISA and to evaluate the 29 kDa and the 31 kDa components of female worm by EITB for serodiagnosis of human angiostrongyliasis. Using ELISA, cross reaction occurred among all sera tested except capillariasis. The sensitivity, specificity, positive and negative predictive values of ELISA were found to be 100%, 66.8%, 27.1% and 100% respectively. Using EITB, The 29 kDa component cross-reacted with most of the heterologous sera while the 31 kDa component was recognized by 69.2% (18/26) sera from angiostrongyliasis patients, cross-reacted only with trichinellosis, trichuriasis and opisthorchiasis sera. This component may possibly used as reliable agents for angiostrongyliasis out of other tissue infected helminthiasis. The sensitivity, specificity, positive and negative predictive values of EITB for the 31.0 kDa component were 69.2%, 82.4%, 46.2% and 92.5% respectively.

Identification of Angiostrongylus cantonensis in infected rats with a coagglutination assay.

The presence of Angiostrongylus cantonensis excretory secretory (ES) products was detected in the cerebrospinal fluid (CSF) of infected rats using a coagglutination assay. There was clear agglutination in 100% of CSF samples tested of the rats infected with third-stage larvae of A. cantonensis and neither of the 20 non-infected rats produced agglutination. The assay detected 250 ng/ml or more of ES products. This assay is simple, easy to perform with minimum training and requires no equipment.

Detection of circulating antigens of Parastrongylus cantonensis in human sera by sandwich ELISA with specific monoclonal antibody.

A specific monoclonal antibody (AW-3C2) as revealed by ELISA was produced against the adult worm antigens of Parastrongylus cantonensis and used in a sandwich ELISA for the detection of circulating antigens in the sera of parastrongyliasis patients and those with other parasitic diseases. A total of 60 sera was used in this study. Of these, 10 each were from patients with parastrongyliasis, cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 serum samples from normal healthy Thais and Malaysians. The mean +/- optical density (OD) values for the normal Thai and Malaysian groups were 0.126 +/- 0.028 and 0.124 +/- 0.029, respectively. The mean OD values of the parastrongyliasis patient group differed significantly from that of the normal groups as well as those of other parasitic infections. Using a cut-off point of OD +/- 3SD of the control groups as indicating a positive reading, the specificity of the assay with this monoclonal antibody was 100% while the sensitivity was 50%.