Neuroimmunological findings of Angiostrongylus cantonensis meningitis in ecuadorian patients

Hallazgos neuroinmunologicos en meningitis por Angiostrongylus cantonesis en pacientes ecuatorianos

ARTICLE

Neuroimmunological findings of Angiostrongylus cantonensis meningitis in ecuadorian patients

Hallazgos neuroinmunologicos en meningitis por Angiostrongylus cantonesis en pacientes ecuatorianos

Alberto J. Dorta-Contreras^I; Barbara Padilla-Docal^I; Juan M. Moreira^II; Luiggi Martini Robles^III; Jenny Muzzio Aroca^III; Fernando Alarcón^IV; Raisa Bu-Coifiu- Fanego^I

^ICentral Spinal Fluid Laboratory (LABCEL), "Dr. Miguel Enríquez" School of Medical Sciences, Havana University of Medical Sciences, Havana, Cuba

^IIDepartment of Public Health Control and Improvement, Ministry of Public Health, Quito, Ecuador

III"Dr. Leopoldo Izquieta Perez" National Hygiene Institute, Guayaquil, Ecuador

^IVNeurology Service, "Eugenio Espejo" Hospital, Quito, Ecuador

^{Correspondence}

ABSTRACT

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% and 26% 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.

Key words:Angiostrongylus cantonensis, Ecuador, eosinophilia, intrathecal synthesis, meningitis, Reibergram.

RESUMEN

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% 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.

Palabras-clave:Angiostrongylus cantonensis, Ecuador, eosinofilia, síntesis intratecal, meningitis, Reibergrama.

Angiostrongylus cantonensis is a zoonotic pathogen that occasionally causes human eosinophilic meningitis. Humans acquire the disease after ingesting intermediate or paratenic hosts such as terrestrial or aquatic molluscs with little or no cooking, or vegetables contaminated by infective third-stage larvae¹.

Angiostrongyliasis is diagnosed on the basis of clinical manifestations, including the presence of cerebrospinal fluid (CSF) eosinophils, and a history of exposure to the larvae. The serological tests vary in sensitivity, and their availability is limited. Therefore, their clinical use is limited.

Angiostrongylus cantonensis meningoencephalitis was first reported in the western hemisphere in Cuba in 1981^1,2. It has subsequently been reported in several Caribbean countries and in the United States^3-9.

In Cuba, human angiostrongyliasis frequently occurs in outbreaks, with numbers of cases that may range from 8 to 100 cases² during rainy periods.

So far, there have not been any reports of neuroimmunological studies on the disease in South America, although the presence of the molluscs, which are intermediate hosts in the lifecycle of the parasite, has been reported in Brazil^10,11, and cases of the disease have been reported in Brazil^12,13 and Ecuador¹⁴.

Currently, the onset of this disease in the western hemisphere is peculiar^15,16.

This paper had the aim of presenting neuroimmunological data on such patients in Ecuador¹⁷.

METHOD

Patients and collection of samples

Eight patients were studied according the infection sources. They were hospitalized at the Neurological Service of the Eugenio Espejo Hospital, Quito, between December 2008 and April 2009. The patients became infected after eating raw (uncooked) molluscs that they had either found in an uncultivated field or bought at an informal market in Quito¹⁷. The diagnosis of A. cantonensis meningitis was based on the history of eating raw molluscs, the symptoms, presence of eosinophils in cerebrospinal fluid (CSF)¹⁸ and the characteristics of the outbreak, which include infected mollusks, infected rats and the presence of third-stage larvae of A. cantonensis in CSF and brain tissue from the outbreak index case who died¹⁷.

The research was approved by the Research Bioethics Committee of Eugenio Espejo Hospital. All the patients gave their informed consent for a diagnostic lumbar puncture procedure to be performed and for their clinical data history to be used.

Simultaneous samples of serum and CSF were collected in order to perform the neuroimmunological analysis. These samples were collected seven days after the onset of the symptoms and small quantities were kept at –80ºC until the time of their use.

CSF and serum analysis

The levels of the major immunoglobulins in serum were measured by means of radial immunodiffusion in NOR Partigen^© plates (Siemens, Marburg) and in CSF using LC Partigen plates (also from Siemens).

Serum albumin was quantified by means of radial immunodiffusion plates in NOR Partigen and albumin in CSF, in LC Partigen^© plates (Siemens, Marburg) in order to determine whether there was any intrathecal synthesis of immunoglobulins and to ascertain the situation of the blood-CSF barrier.

Both the immunoglobulin and the albumin concentration in quotient CSF/serum form were plotted in a Reibergram, which is a CSF/serum quotient diagram for major immunoglobulin classes^18,19.

RESULTS

The eight patients reported in these outbreaks were young adults and belonged to two different families with an average age of 23 years that simultaneously became sick. Headache, vomiting, diarrhea, neck stiffness and severe radicular pain were the most common clinical manifestations, and they started one to two weeks after eating raw molluscs. Two cases showed severe consciousness deficits and one of these entered into a coma. No seizures were reported; imaging findings were negative; and there was no skin damage.

Mean eosinophilia of 7.5% and 26% respectively was observed in the serum and in CSF (Table 1). A moderate increase in proteins was observed in all cases. Glucose levels were normal. The CSF samples from each patient were stained using the Gram stain and the results were negative. The BAAR test, China ink test and microbiological cultures were all negative for the eight patients.

The exclusion criteria that aimed to eliminate other possible causes of angiostrongyliasis included any histories of raw fish consumption, histories of migratory swelling, clinical diagnoses of subarachnoid hemorrhage or myeloencephalitis, positive serological tests for gnathostomiasis or cysticercosis, abnormal brain computed tomography or magnetic resonance findings, symptomatic or serologically positive HIV infection, and active or previous histories of tuberculosis or malignancy. Thus, neurocysticercosis, paragonimiasis, echinococcosis, gnathostomiasis, neurotuberculosis, neuroaids and neuro-coccidioides were discarded.

Table 2 shows the patterns of intrathecal synthesis of major immunoglobulins with their frequencies of appearance per patient. Pattern I was found to be the most frequent type.

Figure shows the Reibergram for major classes of immunoglobulins. All the patients showed intrathecal synthesis of at least two classes of immunoglobulins and all showed intrathecal synthesis of IgM. A three- immunoglobulin intrathecal synthesis pattern is the most frequent one.

DISCUSSION

In Ecuador, it is very common to eat raw molluscs. They are mostly prepared as seviche, which is a spiced dish of raw fish or molluscs marinated in lemon juice, salt and onion. There had never been any reports of transmission of A. cantonensis in Ecuador up to mid-2008. However, several cases of meningoencephalitis in Los Rios and Guayas provinces were then found and, in one of them, the presence of an L5 larva in the subarachnoid space of a dead patient confirmed that the disease was presence and was being transmitted in Ecuador^14,17, with a large parasite load. From that moment on, every outbreak of eosinophilic meningoencephalitis with antecedents of consumption of raw molluscs came to be considered related to A. cantonensis.

Most mollusc species are sensitive and able to transmit the disease. Terrestrial and aquatic molluscs are the primary intermediate hosts¹⁷. In certain Ecuadorian regions, one or two species are intermediate hosts and the infection intensity is very high. The existence of Achatina fulica has been reported in Brazil, and in other countries in South America, which makes it possible for this nematode to be present in the Ecuadorian regions^10,11, with reported cases^12,13. This invertebrate has also been found in large populations in all the places where the disease was reported after the first outbreak¹⁷.

Mollusc species infected with the infective larvae of A. cantonensis were found in Los Rios in Vaginulus sp., Pomacea lineata, Pomacea sp. and Achatina fulica. Regarding the definitive hosts for this parasite, it was found that predominant species infected by the nematode were Rattus rattus and Rattus norvegicus^14,17.

It is thought that A. cantonensis has only recently been introduced into Ecuador and that it was probably caused by infected rats from ships or infected snails.

There were no fatal cases among the eight patients studied, although two of them showed severely impaired consciousness and one of them entered into a coma. However, considering all the notified outbreaks in Ecuador, a total of 26 patients have been diagnosed over a one-year period, of whom eight had neurological sequelae and two died^14,17.

The aggressiveness of the illness when large quantities of third-stage larvae of the parasite are eaten is commonly observed in China, Thailand and other countries in southeastern Asia that have similar nutritional habits^20,21.These sequelae have not been observed in Cuban patients because larva ingestion is purely accidental²².

Dysfunction of the blood-CSF barrier was not a distinctive sign in the patients of this study, since only 37% of the patients involved presented dysfunction of the barrier. This matches with observations from studies in which a second lumbar puncture was performed on patients eight days after the onset of symptoms^22-24.

The observation of the patterns of intrathecal synthesis of immunoglobulins in these Ecuadorian patients is interesting because IgM was present in all of them.

The reported percentage of IgM in Cuban patients was 86%²², while in Ecuadorians it was 100%, but with a 5% general lethality rate (taking the eight patients of the present report into account) and a sequela rate of 22.5%^14,16.

It is likely that infestation caused by A. cantonensis may shortly be reported in other Latin American countries like Peru and Colombia, which share similar habitats and culinary customs. For this reason, the observations of the present study may be of use for the sanitary authorities when dealing with epidemic outbreaks similar to the ones in Ecuador.

These neuroimmunological findings provide further information about the immune response of eosinophilic meningitis caused by A. cantonensis, which is currently a disease that is spreading through South America.

Received 19 July 2010

Received in final form 15 March 2011

Accepted 25 March 2011

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Publication Dates

• Publication in this collection
  19 July 2012
• Date of issue
  June 2011

History

• Reviewed
  15 Mar 2011
• Received
  19 July 2010
• Accepted
  25 Mar 2011