Producción y evaluación del antígeno recombinante TES-30 de Toxocara canis para el inmunodiagnóstico de toxocariasis / Production and evaluation of the recombinant antigen TES-30 of Toxocara canis for the immunodiagnosis of toxocariasis

Introducción. Toxocara canis es un nematodo patógeno de cánidos que accidentalmente puede ser transmitido a los humanos. A pesar de la importancia de la serología para el diagnóstico de esta zoonosis, los kits diagnósticos usan antígenos crudos de excreción-secreción, en su mayoría glucoproteínas que no son específicas de especie, por lo cual pueden presentarse reacciones cruzadas con anticuerpos generados contra otros parásitos. Objetivos. Producir el antígeno recombinante TES-30 de T. canis y evaluarlo para el inmunodiagnóstico de la toxocariasis. Materiales y métodos. Se clonó el gen que codifica TES-30 en el vector de expresión pET28a (+), usando oligonucleótidos de cadena sencilla unidos mediante reacción en cadena de la polimerasa (PCR). La proteína rTES-30 se purificó por cromotografia de afinidad (Ni 2+ ). La reacción serológica de rTES-30 se evaluó mediante immunoblot . Teniendo en cuenta que no existe una prueba de referencia , se observó el comportamiento del antigeno en comparación con la prueba de rutina para el inmunodiagnóstico de la toxocariasis, es decir, la técnica ELISA convencional con antígenos de excreción-secreción. Resultados. El rTES-30 se produjo a partir de un cultivo de Escherichia coli LB, con un rendimiento de 2,25 mg/l y 95 % de pureza. La concordancia de la reacción entre el immunoblot rTES-30 y la ELISA convencional, fue de 73 % (46/63) y de 100 % con los 21 sueros no reactivos. De los 21 sueros con diagnóstico de otras parasitosis, 19 fueron reactivos con ELISA, mientras que tan solo siete fueron positivos con el immunoblot rTES-30. La concordancia entre la ELISA y el immunoblot fue moderada (índice kappa de 0,575; IC 95% 0,41-0,74). Conclusiones. Los datos presentados respaldan la utilidad del immunoblot r TES-3 0 para la confirmación de los posibles positivos por ELISA, no solo en los estudios epidemiológicos, sino también, como candidato para el desarrollo de pruebas diagnósticas de la toxocariasis ocular en Colombia.

Introduction: Toxocara canis is a pathogenic nematode of canines which can be accidentally transmitted to humans. Although serology is the most important diagnostic tool for this zoonosis, diagnostic kits use crude excretion/secretion antigens, most of them being glycoproteins which are not species-specific and may cross-react with antibodies generated against other parasites. Objectives: To produce the rTES-30 recombinant antigen of Toxocara canis and evaluate it in the immunodiagnosis of toxocariasis. Materials and methods: The gene that codes for TES-30 was cloned in the expression vector pET28a (+) using single-stranded oligonucleotides united by PCR. The protein rTES-30 was purified by Ni 2+ affinity chromotography. Seroreactivity of rTES-30 was evaluated by immunoblot. Given that there is no gold standard test, the behaviour of the antigen was compared with the method that is routinely used to immunodiagnose toxocariasis, i.e., the conventional ELISA technique using excretion/secretion antigens. Results: The rTES-30 was produced from an Escherichia coli LB culture which yielded 2.25 mg/L of the antigen with a purity of 95%. The results obtained showed 73% (46/63) concordance of reactivity between the rTES-30 immunoblot and the conventional ELISA, and 100% concordance with the non-reactive sera (21). Nineteen of the 21 sera positive for other parasitoses reacted with ELISA, while only seven of these were positive with the rTES-30 immunoblot. Concordance between the ELISA and the immunoblot was moderate (kappa coefficient: 0.575; 95% CI: 0.41- 0.74). Conclusions: The data presented show the potential of the rTES-30 inmunoblot for confirmation of possible ELISA positives, not only in epidemiological studies, but also as a candidate for the development of diagnostic tests for ocular toxocariasis in Colombia.

Kinetics and avidity of anti-Toxocara antibodies (IgG) in rabbits experimentally infected with Toxocara canis / Cinética e avidez de anticorpos (IgG) anti-Toxocara em coelhos experimentalmente infectados com Toxocara canis

Abstract An evaluation was made of the kinetics and avidity of anti-Toxocara antibodies (IgG) in rabbits experimentally infected with embryonated Toxocara canis eggs. Seventeen four month old New Zealand White rabbits were distributed into two groups. In the experimental group, twelve rabbits were infected orally with 1,000 embryonated T. canis eggs. A second group (n = 5), uninfected, was used as a control. Serum samples were collected for analysis on days 7, 14, 21, 28 and 60 post-infection (DPI). An indirect ELISA test was performed to evaluate the reactivity index (RI) of IgG anti-T. canis antibodies and to calculate the avidity index (AI). The animals showed seroconversion from the 14th DPI, with high AI (over 50%) except for one animal, which presented an intermediate AI. At 60 DPI, all the animals were seropositive and maintained a high AI. The data indicated that specific IgG antibodies formed early (14 DPI) in rabbits infected with T. canis, with a high avidity index that persisted throughout the course of the infection.

Resumo O objetivo deste estudo foi o de avaliar a cinética e a avidez de anticorpos anti-Toxocara canis, em coelhas infectadas experimentalmente com ovos embrionados de Toxocara canis. Foram utilizados 17 coelhos New Zealand de linhagem branca, com quatro meses de idade, distribuídos em dois grupos. No grupo experimental, doze coelhas foram infectadas, oralmente, com 1.000 ovos larvados de T. canis. Um segundo grupo (n=5), não infectado, foi utilizado como controle. Nos dias 7, 14, 21, 28 e 60 pós-infecção (DPI), foram coletadas amostras de soro para análise. O teste de ELISA indireto foi realizado para avaliar o índice de reatividade (IR) de anticorpos IgG anti-T. canis e para cálculo do índice de avidez (IA). A soroconversão nos animais ocorreu a partir do140 DPI, com verificação de alto IA (superior a 50%), com exceção de um animal, que apresentou médio IA. Aos 60 DPI, todos os animais foram soropositivos e mantiveram alto IA. Os dados mostram que em coelhos infectados por T. canis, anticorpos IgG específicos formam-se precocemente (14 DPI), apresentando alto índice de avidez e que se mantém durante o curso da infecção.

Diagnostic Value of the Serum Anti-Toxocara IgG Titer for Ocular Toxocariasis in Patients with Uveitis at a Tertiary Hospital in Korea

PURPOSE: This study evaluated the prevalence of ocular toxocariasis (OT) in patients with uveitis of unknown etiology who visited a tertiary hospital in South Korea and assessed the success of serum anti-Toxocara immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) as a diagnostic test for OT. METHODS: The records of consecutive patients with intraocular inflammation of unknown etiology were reviewed. All participants underwent clinical and laboratory investigations, including ELISA for serum anti-Toxocara IgG. OT was diagnosed based on typical clinical findings. Clinical characteristics, seropositivity, and IgG titers were compared between patients diagnosed with OT and non-OT uveitis. The seropositivity and the diagnostic value of anti-Toxocara IgG was investigated among patients with different types of uveitis. RESULTS: Of 238 patients with uveitis of unknown etiology, 71 (29.8%) were diagnosed with OT, and 80 (33.6%) had positive ELISA results for serum anti-Toxocara IgG. The sensitivity and specificity of the ELISA test were 91.5% (65 / 71) and 91.0% (152 / 167), respectively. The positive predictive value of the serum anti-Toxocara IgG assay was 81.3%. Among patients with anterior, intermediate, posterior, and panuveitis, the prevalence rates of OT were 8.3%, 47.1%, 44.8%, and 7.1%, respectively; the seropositivity percentages were 18.1%, 47.1%, 43.7%, and 17.9%; and the positive predictive values were 38.5%, 95.8%, 92.1%, and 40.0%. The serum anti-Toxocara IgG titer also significantly decreased following albendazole treatment. CONCLUSIONS: OT is a common cause of intraocular inflammation in the tertiary hospital setting. Considering that OT is more prevalent in intermediate and posterior uveitis, and that the positive predictive value of the anti-Toxocara IgG assay is high, a routine test for anti-Toxocara IgG might be necessary for Korean patients with intermediate and posterior uveitis.

Toxocara canis and the allergic process

The protective effect of infectious agents against allergic reactions has been thoroughly investigated. Current studies have demonstrated the ability of some helminths to modulate the immune response of infected hosts. The objective of the present study was to investigate the relationship between Toxocara canis infection and the development of an allergic response in mice immunised with ovalbumin (OVA). We determined the total and differential blood and bronchoalveolar lavage fluid cells using BALB/c mice as a model. To this end, the levels of interleukin (IL)-4, IL-5 and IL-10 and anti-OVA-IgE were measured using an ELISA. The inflammatory process in the lungs was observed using histology slides stained with haematoxylin and eosin. The results showed an increase in the total number of leukocytes and eosinophils in the blood of infected and immunised animals at 18 days after infection. We observed a slight lymphocytic inflammatory infiltrate in the portal space in all infected mice. Anti-OVA-IgE levels were detected in smaller proportions in the plasma of immunised and infected mice compared with mice that were only infected. Therefore, we concluded that T. canis potentiates inflammation in the lungs in response to OVA, although anti-OVA-IgE levels suggest a potential reduction of the inflammatory process through this mechanism.

Seropositivity for ascariosis and toxocariosis nd cytokine expression among the indigenous people in the Venezuelan delta region / Seropositividad para ascariosis y toxocariosis y expresión de citocinas entre la población indígena de la región del delta Venezolano

The present study aimed at measuring seropositivities for infection by Ascaris suum and Toxocara canis using the excretory/secretory (E/S) antigens from Ascaris suum (AES) and Toxocara canis (TES) within an indigenous population. In addition, quantification of cytokine expressions in peripheral blood cells was determined. A total of 50 Warao indigenous were included; of which 43 were adults and seven children. In adults, 44.1% were seropositive for both parasites; whereas children had only seropositivity to one or the other helminth. For ascariosis, the percentage of AES seropositivity in adults and children was high; 23.3% and 57.1%, respectively. While that for toxocariosis, the percentage of TES seropositivity in adults and children was low; 9.3% and 14.3%, respectively. The percentage of seronegativity was comparable for AES and TES antigens in adults (27.9%) and children (28.6%). When positive sera were analyzed by Western blotting technique using AES antigens; three bands of 97.2, 193.6 and 200.2 kDas were mostly recognized. When the TES antigens were used, nine major bands were mostly identified; 47.4, 52.2, 84.9, 98.2, 119.1, 131.3, 175.6, 184.4 and 193.6 kDas. Stool examinations showed that Blastocystis hominis, Hymenolepis nana and Entamoeba coli were the most commonly observed intestinal parasites. Quantification of cytokines IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 and IL-4 expressions showed that there was only a significant increased expression of IL-4 in indigenous with TES seropositivity (p < 0.002). Ascaris and Toxocara seropositivity was prevalent among Warao indigenous.

El objetivo del presente estudio fue determinar la seropositividad de infección por Ascaris suum y Toxocara canis, utilizando antígenos de excreción/secreción (E/S) de Ascaris suum (AES) y Toxocara canis (TES) en una población indígena. Adicionalmente, se cuantificó la expresión de citocinas a partir de células de sangre periférica. Un total de 50 indígenas Warao se incluyeron en el estudio; 43 fueron adultos y 7 niños. Entre los adultos, 44,1% fueron seropositivos para ambos parásitos; mientras que los niños sólo mostraron seropositividad a uno u otro de los helmintos. Para ascariosis, el porcentaje de seropositividad para los antígenos AES fue alto tanto en adultos como en niños; 23,3% y 57,1%, respectivamente. Para toxocariosis, el porcentaje de seropositividad para los antígenos TES fue bajo en adultos así como en niños; 9,3% y 14,3%, respectivamente. El porcentaje de seronegatividad fue similar tanto para los antígenos AES como para TES en adultos (27,9%) y niños (28,6%). Cuando la seropositividad fue analizada a través de la técnica de Western blotting utilizando los antígenos AES; 3 bandas de 97,2, 193,6 y 200,2 kDas fueron principalmente reconocidas. Para los antígenos TES, 9 bandas fueron mayormente identificadas; 47,4, 52,2, 84,9, 98,2, 119,1, 131,3, 175,6, 184,4 y 193,6 kDas. Los análisis coproparasitológicos mostraron que los parásitos Blastocystis hominis, Hymenolepis nana y Entamoeba coli fueron los parásitos intestinales más comúnmente observados. La cuantificación de la expresión de las citocinas IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 e IL-4 mostró que hubo un significante incremento de la expresión de IL-4 entre los indígenas con seropositividad para los antígenos TES (p < 0.002). La seropositividad para Ascaris y Toxocara fue prevalente entre los indígenas Warao.

Immunopathological Changes in the Brain of Immunosuppressed Mice Experimentally Infected with Toxocara canis

Toxocariasis is a soil-transmitted helminthozoonosis due to infection of humans by larvae of Toxocara canis. The disease could produce cognitive and behavioral disturbances especially in children. Meanwhile, in our modern era, the incidence of immunosuppression has been progressively increasing due to increased incidence of malignancy as well as increased use of immunosuppressive agents. The present study aimed at comparing some of the pathological and immunological alterations in the brain of normal and immunosuppressed mice experimentally infected with T. canis. Therefore, 180 Swiss albino mice were divided into 4 groups including normal (control) group, immunocompetent T. canis-infected group, immunosuppressed group (control), and immunosuppressed infected group. Infected mice were subjected to larval counts in the brain, and the brains from all mice were assessed for histopathological changes, astrogliosis, and IL-5 mRNA expression levels in brain tissues. The results showed that under immunosuppression, there were significant increase in brain larval counts, significant enhancement of reactive gliosis, and significant reduction in IL-5 mRNA expression. All these changes were maximal in the chronic stage of infection. In conclusion, the immunopathological alterations in the brains of infected animals were progressive over time, and were exaggerated under the effect of immunosuppression as did the intensity of cerebral infection.

Immunopathological Changes in the Brain of Immunosuppressed Mice Experimentally Infected with Toxocara canis

Toxocariasis is a soil-transmitted helminthozoonosis due to infection of humans by larvae of Toxocara canis. The disease could produce cognitive and behavioral disturbances especially in children. Meanwhile, in our modern era, the incidence of immunosuppression has been progressively increasing due to increased incidence of malignancy as well as increased use of immunosuppressive agents. The present study aimed at comparing some of the pathological and immunological alterations in the brain of normal and immunosuppressed mice experimentally infected with T. canis. Therefore, 180 Swiss albino mice were divided into 4 groups including normal (control) group, immunocompetent T. canis-infected group, immunosuppressed group (control), and immunosuppressed infected group. Infected mice were subjected to larval counts in the brain, and the brains from all mice were assessed for histopathological changes, astrogliosis, and IL-5 mRNA expression levels in brain tissues. The results showed that under immunosuppression, there were significant increase in brain larval counts, significant enhancement of reactive gliosis, and significant reduction in IL-5 mRNA expression. All these changes were maximal in the chronic stage of infection. In conclusion, the immunopathological alterations in the brains of infected animals were progressive over time, and were exaggerated under the effect of immunosuppression as did the intensity of cerebral infection.

Enhanced Resolution of Eosinophilic Liver Abscess Associated with Toxocariasis by Albendazole Treatment / 대한소화기학회지

BACKGROUND/AIMS: Visceral larva migrans, caused by Toxocara canis and Toxocara cati, has emerged as a significant cause of eosinophilic liver abscess (ELA). Differentiation of ELA associated with toxocariasis (ELA-T) from metastasis or primary liver malignancy is sometimes difficult. However, the role of albendazole treatment remains uncertain in this condition. The aim of this study was to evaluate whether albendazole can enhance the radiologic resolution of ELA-T. METHODS: We retrospectively reviewed the medical records of the patients diagnosed with ELA-T at our institution between January 2008 and December 2011. ELA-T was diagnosed based on the imaging findings on computed tomography or magnetic resonance imaging and the presence of positive serum IgG antibody for Toxocara canis. Among a total of 163 patients, 32 patients received albendazole (albendazole group) and 131 did not (control group). Baseline characteristics and fate of liver nodules were compared between the two groups. RESULTS: Baseline characteristics (age, sex, number and maximal size of lesions, eosinophil count) were similar between the two groups. Median duration for achieving radiologic resolution in the albendazole group was significantly shorter than in the control group (207 days [range 186-228] vs. 302 days [range 224-380], p=0.023). In Cox regression analysis of the cumulative rates of radiologic resolution, the hazard ratio for albendazole treatment was 1.99 (95% confidence interval, 1.22-3.23). CONCLUSIONS: Radiologic resolution of ELA-T can be accelerated with albendazole treatment. Hence, inconvenience associated with long-term follow-up and unnecessary worries among patients can be eliminated with albendazole treatment.

Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis / Determinação da avidez de IgG em camundongos BALB/c experimentalmente infectados com Toxocara canis

Toxocariasis is a zoonotic disease in that IgM titers can remain high for long periods making difficult to determine the stage of the disease. The aim of this study is to investigate the applicability of indirect ELISA, associated with urea, to discriminate between the acute and chronic toxocariasis. IgG avidity was evaluated in 25 BALB/c mice experimentally infected with 1000 Toxocara canis eggs. Blood samples were collected, and sera treated with 6 M urea and assayed by ELISA every two weeks. The percent IgG avidity was determined using the mean absorbance of sera treated with urea, divided by the mean absorbance of untreated sera. In the first 15 days post-inoculation, was observed a low percentage, between 7.25 and 27.5%, IgG avidity, characteristic of an acute infection. After 60 days of infection, all the mice showed between 31.4 and 58% IgG avidity, indicating a chronic infection.

A toxocaríase é uma zoonose na qual os títulos de IgM podem permanecer elevados por longos períodos, tornando difícil a determinação do estágio em que a doença se encontra. O objetivo deste estudo foi investigar a aplicabilidade de um teste indireto de ELISA, associado com ureia, para fazer a discriminação entre as fases aguda e crônica da toxocaríase. A avidez de IgG foi avaliada em 25 camundongos BALB/c experimentalmente infectados com 1000 ovos embrionados de Toxocara canis. A cada duas semanas, amostras de sangue foram coletadas, o soro tratado com ureia 6M e realizado o ensaio pela técnica de ELISA. O percentual de avidez de IgG foi determinado, usando-se a média das absorbâncias dos soros tratados com ureia dividida pela média das absorbâncias dos soros não tratados. Nos primeiros 15 dias pós-inoculação, foi observado um baixo percentual de avidez de IgG, entre 7,25 e 27,5%, característico da fase aguda da infecção. Após 60 dias de infecção, todos apresentaram avidez de IgG entre 31,4 e 58%, indicando a fase crônica da infecção.

Toxocariasis: seroprevalence in abandoned-institutionalized children and infants / Toxocariosis: seroprevalencia en infantes expósitos, abandonados e institucionalizados

Toxocariasis is an infection that has worldwide distribution. Toxocara canis is the most relevant agent due to its frequent occurrence in humans. Soil contamination with embryonated eggs is the primary source of T. canis. This study aimed to determine the seroprevalence of toxocariasis in 10-month to 3 year-old abandoned infants, considered to be at high risk because of their orphanhood status and early age. Blood samples were collected from 120 children institutionalized in an orphanage in the city of La Plata. In this study, we observed 38.33% of seropositive cases for T. canis by ELISA and 45% by Western blot techniques; significant differences among groups A (<1 year), B (1-2 years) and C (>2 years) were also found. In research group A, children presented a seropositivity rate of 23.91%, in group B of 42.85% and in group C of 56%, which indicates an increase in frequency as age advances, probably because of greater chances of contact with infective forms of the parasite since canines and soil are frequently infected with T. canis eggs. Abandoned children come from poor households, under highly unsanitary conditions resulting from inadequate or lack of water supply and sewer networks, and frequent promiscuity with canines, which promotes the occurrence of parasitic diseases. These children are highly vulnerable due to their orphanhood status and age.

La toxocariosis es una enfermedad presente en todo el mundo. Como causa primaria de infección se cita la contaminación de los suelos con huevos embrionados de Toxocara canis. Nuestro objetivo fue determinar la seroprevalencia de toxocariosis en niños expósitos (abandonados) de 10 meses hasta 3 años, los que se consideran de alto riesgo por su condición de orfandad y escasa edad. Las muestras de sangre fueron recolectadas de 120 niños institucionalizados en un orfelinato de la ciudad de La Plata. En este estudio, se observó un porcentaje de seropositivos para T. canis de 38,33 % por la técnica de ELISA y de 45 % por la técnica de Western blot, con diferencias significativas entre los grupos etarios estudiados (A: < 1 año, B: 1-2 años, C: > 2 años). Los niños del grupo A presentaron una frecuencia de seropositividad de 23,91 %; los del grupo B, de 42,85 % y en los niños del grupo C fue del 56 %. Esto indica un incremento de la frecuencia de presentación a medida que aumentó la edad, debido probablemente a las mayores posibilidades de contactar con estados infectantes del parásito, ya que los caninos y el suelo se hallan frecuentemente infectados por huevos de T. canis. Los niños abandonados provienen de hogares carenciados, donde a las malas condiciones de higiene resultantes de la ausencia de red de agua y cloacal se le agrega la frecuente promiscuidad con caninos, lo cual propicia la presencia de parasitosis. Sumado a la condición de desamparo, esto produce un estado de máxima vulnerabilidad.

Isolation of IgG Antibodies to Toxocara in Ankylosing Spondylitis Patients with Acute Anterior Uveitis

PURPOSE: Since few reports had been published on the prevalence of toxocariasis in ankylosing spondylitis (AS) patients with acute non-granulomatous anterior uveitis (ANGAU), the aim of this work was to determine the presence of antibodies against Toxocara canis in AS patients with ANGAU. METHODS: Thirty-six patients (14 female and 22 male) with AS were enrolled in the study. The history of ANGAU was accepted only if diagnosed by an ophthalmologist. The detection of IgG antibodies to T. canis was determined by enzyme-linked immunosorbent assay. In addition, antibodies to Ascaris lumbricoides were also tested to verify non-specific reactions. RESULTS: The prevalence of ANGAU in the AS patients was 58% (21 / 36), and 38% (8 / 21) of the patients with ANGAU were positive for antibodies to Toxocara, while 7% (1 / 15) of AS patients without ANGAU were positive for T. canis (p = 0.038, two tails; mid-p exact). No antibodies were detected to A. lumbricoides antigens in the serum samples of patients with AS. CONCLUSIONS: These data suggest that the seroprevalence of antibodies to T. canis is high in Mexican patients with AS-associated uveitis, suggesting a chronic asymptomatic toxocariosis, which could be associated with the pathogenesis of ANGAU; however, further larger-scale studies are needed to confirm this observation.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites / Anticorpos IgG em camundongos coinfectados por Toxocara canis e outros helmintos e por protozoários parasitos

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

Estudou-se a resposta imune humoral expressa por anticorpos da classe IgG em camundongos BALB/c experimentalmente infectados com Toxocara canis em duas situações distintas. Na primeira, com o objetivo de verificar in vivo a possível reatividade cruzada entre Toxocara canis e outros parasitos (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis e Toxoplasma gondii), foram realizados experimentos constituídos por três grupos de camundongos: um infectado apenas por Toxocara canis, outro com uma das demais espécies de parasitos estudados e um terceiro concomitantemente infectado por Toxocara canis e a espécie em questão. Todos os animais foram sangrados, através do plexo orbitário, 23, 38 e 70 dias após infecção. Os soros foram analisados para a presença de anticorpos anti-Toxocara por meio de teste imunoenzimático (ELISA) e por Immunoblotting, empregando-se antígeno de excreção-secreção (ES), obtido a partir da cultura de larvas de terceiro estádio de Toxocara canis. Para todos os experimentos utilizou-se grupo controle negativo constituído por 10 camundongos não infectados. Apenas no caso da infecção por Ascaris suum, nas condições experimentais observadas, logrou-se demonstrar ocorrência de reatividade cruzada com antígenos de Toxocara canis. Verificou-se, entretanto, no caso das coinfecções entre Toxocara canis-Schistosoma mansoni, Toxocara canis-Strongyloides venezuelensis e Toxocara canis-Taenia crassiceps produção de anticorpos anti-Toxocara em níveis significativamente inferiores do que os encontrados nos camundongos infectados somente por Toxocara canis. Nas coinfecções com Schistosoma mansoni ou Strongyloides venezuelensis observou-se, também, menor taxa de letalidade quando comparada à ocorrida nos animais com as respectivas infecções simples.

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85 percent and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Achados ultrassonográficos em toxocaríase ocular / Ultrasonographic findings in ocular toxocariasis

OBJETIVO: Avaliar achados ultrassonográficos nas três principais formas de apresentação da toxocaríase ocular (granulomas de periferia e polo posterior e endoftalmite crônica), em pacientes com diagnóstico confirmado de toxocaríase ocular. MÉTODOS: Foram analisados no estudo 11 pacientes (11 olhos), de forma prospectiva, com diagnóstico de toxocaríase forma ativa, com teste ELISA positivo. Os pacientes foram submetidos ao exame de ultrassonografia ocular (transdutor 10 MHz, técnica de contato). RESULTADOS: Na série de 11 pacientes, com idade média de 7,9 anos (variando de 2 a 17 anos), 73% homens, referiram contato prévio com cães (91%), e com solo (50%), sem referência à perversão do apetite. Na avaliação dos olhos comprometidos (11 olhos), o exame oftalmológico revelou a seguinte distribuição das três formas de toxocaríase ocular: 7 (63,6%), granuloma de polo posterior; 1 (9,1%), endoftalmite crônica; 2 (18,2%), granuloma periférico; e 1 (9,1%), quadro associado de granuloma de polo posterior e endoftalmite crônica. Acuidade visual comprometida: sem percepção luminosa (3 olhos, 27,3%); visão de vultos (4 olhos, 36,4%); contar dedos a 10 cm (1 olho, 9,1%); 20/200 (1 olho, 9,1%); 20/70 (1 olho, 9,1%); indeterminado (1 olho, 9,1%). Sorologia para Toxocara canis foi positiva (teste ELISA) em 100% dos casos. Oftalmoscopia foi difícil ou impossível em 64% dos casos devido à opacidade de meios. Características ultrassonográficas observadas: membranas vítreas com retina aplicada (100%); lesões de parede (granulomas) com refletividade alta (80%) ou média (20%). CONCLUSÃO: O achado ultrassonográfico mais consistente no olho portador de toxocaríase foi a presença de massa retiniana de alta refletividade, localizada no polo posterior ou periferia, que pode ser calcificada, e que apresenta como principal característica a aderência de membranas vítreas. Em combinação com a história, exame clínico e sorologia, a ultrassonografia pode ajudar no diagnóstico da toxocaríase ocular, principalmente nos casos com opacidade de meios.

PURPOSE: To evaluate ophthalmic ultrasound findings in the three presentation forms of ocular toxocariasis (peripheral or posterior pole granulomas and chronic endophthalmitis), in patients with confirmed diagnosis of ocular toxocariasis. METHODS: 11 patients (11 eyes) with clinical and confirmed diagnosis of active ocular toxocariasis, presented positive ELISA test, were analyzed, prospectively, in the study. The patients were submitted to an ocular ultrasound examination (10-MHz transducer, contact technique). RESULTS: In the series of 11 patients, mean age was 7.9 years-old (range from 2 to 17 y), 73% male, referring previous contact with dogs (91%), and with the soil (50%), no referral of appetite perversion. In the analyses of compromised eyes (11 eyes), the ophthalmoscopic examination revealed the following distribution of the 3 forms of ocular toxocariasis: 7 cases (63.6%), posterior pole granuloma; 1 (9.1%), chronic endophthalmitis; 2 (18.2%), peripheral granuloma; and 1 (9.1%), posterior pole granuloma associated with chronic endophthalmitis. Visual acuity impairment: no light perception (3 eyes, 27.3%); hand motion (4 eyes, 36.4%); counting fingers at 10 cm (1 eye, 9.1%); 20/200 (1 eye, 9.1%); 20/70 (1 eye, 9.1%); undefined (1 eye, 9.1%). Serology was positive to Toxocara canis (ELISA test) in 100% of the cases. Ophthalmoscopy was difficult or impossible in 64% of the cases due to the media opacity. Ultrasound findings noted were vitreous membranes with retinal attachment (100%); parietal lesions (granulomas) with high (80%) or medium (20%) reflectivity. CONCLUSION: The most consistent ultrasound finding in the eye with toxocariasis was a high-reflectivity retinal mass, located in posterior pole or periphery, which may be calcified, and which has as main characteristic the adherence of vitreous membranes. In addition to clinical history, systemic evaluation and serology, the ultrasound can help in the diagnosis of ocular toxocariasis, especially in media opacities.

Prevalência e fatores de risco da infecção humana por Toxocara canis em Salvador, Estado da Bahia / Prevalence and risk factors of human infection by Toxocara canis in Salvador, State of Bahia, Brazil

INTRODUÇÃO: Larva migrans visceral é causada por Toxocara sp e nunca foi estudada na Bahia. Neste trabalho, investigou-se a prevalência e fatores de risco de infecção por Toxocara canis, em indivíduos de Salvador. MÉTODOS: Trezentos e trinta e oito indivíduos foram investigados para presença de anticorpos IgG séricos anti-T. canis. RESULTADOS: IgG anti-T. canis foi mais alta em indivíduos de classe social baixa com maior contato com cães e gatos, indicando que estas variáveis são fatores de risco para esta infecção. CONCLUSÕES: A prevalência de infecção por T. canis foi alta. Os fatores de risco desta infecção encontrados estão de acordo com a literatura.

INTRODUCTION: Larva migrans visceral is caused by Toxocara sp and has never been studied in Bahia. This work investigated the prevalence and risk factors for infection by Toxocara canis in individuals from Salvador, State of Bahia. METHODS: Three hundred and thirty-eight individuals were investigated for the presence of serum IgG anti-T. canis. RESULTS: IgG anti-T. canis was higher in individuals from lower social classes who had more contact with dogs and cats, indicating that these variables are factors risk for this infection. CONCLUSIONS: The prevalence of T. canis infection was high. The risk factors for this infection identified are in agreement with in the literature.

Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis / Estudo de marcadores imunológicos para o diagnóstico e avaliação terapêutica da toxocaríase

In human toxocariasis, there are few approaches using immunological markers for diagnosis and therapeutic assessment. An immunoblot (IB) assay using excretory-secretory Toxocara canis antigen was standardized for monitoring IgG, IgE and IgA antibodies in 27 children with toxocariasis (23 visceral, three mixed visceral and ocular, and one ocular form) for 22-116 months after chemotherapy. IB sensitivity was 100 percent for IgG antibodies to bands of molecular weight 29-38, 48-54, 95-116, 121-162, >205 kDa, 80.8 percent for IgE to 29-38, 48-54, 95-121, > 205 kDa, and 65.4 percent for IgA to 29-38, 48-54, 81-93 kDa. Candidates for diagnostic markers should be IgG antibodies to bands of low molecular weight (29-38 and 48-54 kDa). One group of patients presented the same antibody reactivity to all bands throughout the follow-up study; in the other group, antibodies decayed partially or completely to some or all bands, but these changes were not correlated with time after chemotherapy. Candidates for monitoring patients after chemotherapy may be IgG antibodies to > 205 kDa fractions, IgA to 29-38, 48-54, 81-93 kDa and IgE to 95-121 kDa. Further identification of antigen epitopes related to these markers will allow the development of sensitive and specific immunoassays for the diagnosis and therapeutic assessment of toxocariasis.

Métodos imunológicos desempenham papel importante no diagnóstico da toxocaríase, entretanto há poucos estudos sobre marcadores diagnósticos e de acompanhamento terapêutico. Foi padronizado ensaio de immunoblot (IB) empregando antígeno de excreção-secreção de Toxocara canis para pesquisa de anticorpos IgG, IgE e IgA em 27 crianças com toxocaríase nas formas visceral (23), mista visceral e ocular (3) e ocular (1), por 22-116 meses após quimioterapia. Foram observados dois perfis de reatividade dos anticorpos: permanência contra todas as frações no decorrer do estudo; diminuição ou negativação contra algumas ou todas as frações, porém, essas mudanças não se correlacionaram com tempo de tratamento. A sensibilidade do IB foi 100,0 por cento para anticorpos IgG específicos para frações de massa molecular de 29-38, 48-54, 95-116, 121-162, > 205 kDa, 80,8 por cento para IgE específicos para 29-38, 48-54, 95-121, > 205 kDa e 65,4 por cento para IgA específicos para 29-38, 48-54, 81-93 kDa. Anticorpos IgG específicos para frações de baixa MM (29-38 e 48-54 kDa) podem ser sugeridos como candidatos a marcadores diagnósticos. Por sua vez, anticorpos IgG para fração > 205 kDa, IgA para 29-38, 48-54, 81-93 kDa e IgE para 95-121 kDa podem ser candidatos a marcadores terapêuticos. A identificação de epítopos antigênicos relacionados a estes marcadores poderá ser importante para o desenvolvimento de ensaios altamente sensíveis e específicos no diagnóstico e avaliação terapêutica da toxocaríase.

Identification of candidate antigens from adult stages of Toxocara canis for the serodiagnosis of human toxocariasis

In the present work, we identified adult Toxocara canis antigens through sodium dodecyl sulfate-polyacrylamide gel electrophoresis for potential use in human toxocariasis immunodiagnosis. The sensitivity and specificity of several semi-purified antigens, as well as their cross-reactivity with other parasitic infections, were assessed by IgM and IgG-enzime linked immunosorbent assay. Whilst we found that the crude extract of the parasite presented limited sensitivity, specificity and high cross-reactivity against other parasites, we identified 42, 58, 68 and 97-kDa semi-purified antigens as the most promising candidates for immunodiagnosis. Moreover, the 58 and 68-kDa antigens presented the lowest IgM cross-reactivity. When tested as a combination, a mixture of the 58 and 68-kDa antigens presented 100 percent sensitivity and specificity, as well as minor cross-reactivity. Although the combination of the 42, 58, 68 and 97-kDa antigens presented 100 percent sensitivity at a dilution of 1:40, the low specificity and high cross-reactivity observed suggested a limited use for diagnostic purposes. Our data suggested that the 58 and 68-kDa antigens might be most suitable for the immunodiagnosis of human toxocariasis.

Neurotoxocariasis associated with lower motor neuron disease: report of one case / Neurotoxocariasis asociada a enfermedad de motoneurona inferior: presentación de un caso

We report a 51-year-old male with a history of palpitations, hepatopathy and hypercholesterolemia, who habitually ate raw goat meat, and developed general fasciculations, muscle cramps in the lower limbs, distal muscle weakness and wasting, without upper motor neuron signs or sensory abnormalities. Diagnostic workup revealed positive antibodies against Toxocara canis in the serum and cerebrospinal fuid. Nerve conduction studies revealed a proximal and distal axonal lesion of motor nerves and needle electromyography was indicative of acute and chronic denervation with giant motor unit action potentials. Despite a therapy with albendazole and riluzole, muscle weakness and wasting further progressed and affected also the respiratory muscles. Followup nerve conduction studies and electromyography confrmed progression of the axonal degeneration. Whether lower motor neuron disease was causally related to neurotoxocariasis or due to a general metabolic defect, remains speculative.

Presentamos un hombre de 51 años con una historia de palpitaciones, hepatopatía e hipercolesterolemia que comía habitualmente carne de cabra cruda, que desarrolló un cuadro caracterizado por fasciculaciones generalizadas, calambres musculares en las extremidades inferiores, pérdida de fuerza y atrofa muscular distal, sin signos de lesión de motoneurona superior o alteraciones sensitivas. El laboratorio mostró anticuerpos anti Toxocara canis en suero y líquido cefalorraquídeo. La electromiografía mostró una lesión axonal proximal y distal de nervios motores y denervación con potenciales de acción gigantes en las unidades motoras. A pesar de tratarse con albendazol y riluzona, la debilidad muscular y atrofa continuaron progresando. Una nueva electromiografía confrmó la progresión de la degeneración axonal. La asociación entre esta enfermedad de motoneurona inferior y la neurotoxocariasis, es especulativa.

Toxocara canis y asma bronquial / Toxocara canis and bronchial asthma

A fin de evaluar la relación entre la infección por Toxocara canis y los síntomas del asma bronquial en niños de una región subtropical con alta prevalencia de toxocariosis, se estudiaron 47 niños con asma y 53 sin asma como grupo control. Se efectuó el examen físico completo, registrándose datos clínicos y epidemiológicos. En los niños con asma se categorizó el patrón de presentación, frecuencia y gravedad de los síntomas con una escala de I a IV. Se investigó la presencia de anticuerpos anti-Toxocara canis en ambos grupos mediante el método de ELISA en fase sólida, empleando antígeno de excreción/secreción y se efectuó dosaje de Ig E total. Los resultados muestran una seropositividad del 55% en el total de los niños, del 57.4% en los niños con asma y del 52.8% en los controles. En los niños con sintomatología más grave (grado II, III y IV) hubo un 67.7% de seropositivos, mientras que en los niños con síntomas de grado I la seropositividad fue de 37.5% (p = 0.0470). La infección por T. canis actuaría como un co-factor agravante de los síntomas del asma bronquial.

In order to evaluate the association between the infection by Toxocara canis and the symptoms of asthma in children from a subtropical region with high prevalence of toxocariasis, 47 asthmatic children and 53 non-asthmatics as a control group were studied. A complete physical examination was performed and clinical and epidemiological data were registered. In asthmatic children the frequency and severity of symptoms were classified in grades I to IV. The presence of anti-Toxocara canis antibodies in both groups was evaluated employing a solid phase ELISA method with excretion/secretion antigens, and total Ig E was also measured. Results showed a total seropositivity of 55%, 57.4% in children with asthma and 52.8% in the control group. Among asthmatics with severe symptoms (grade II, III and IV), there was a 67.7% of seropositivity while in children with symptoms of grade I there was a 37.5% (p = 0.0470). The infection with T. canis could act as a co-factor increasing the severity of the symptoms of bronchial asthma.

[Frequency of anti toxocara antibodies in schoolchildren with chronic cough and eosinophilia in urban and rural area of Ahvaz, Iran in 2006]

It has been suggested that chronic cough may be atributed to toxocariasis. There are few epidemiological studies carried out on this subject in Iran. The aim of this study was to determine the frequency of anti-toxocara antibodies in children with chronic cough. In a cross sectional study during a tuberculin survey in Ahvaz, a city in southwest Iran a total of 4206 children selected by randomized two-stage cluster sampling. Children with chronic cough were investigated for toxocariasis. Initially, the absolute eosinophil count was determined. Next, children with eosinophilia [>500/mm[3]] and hypereosinophilia [>1000/mm[3]] were tested for toxocara-IgG - ELISA kit [Ibl, Hamburg]. Other epidemiological data collected by a questionnaire and analyzed with 95% confidence interval using SPSS 11.5 software. Of total 115 children with chronic cough 29 [25.2%] were eosinophilic, among them 34.5% were hypereosinophilic, 16 [13.9%] were ELISA-IgG positive for T. canis. No correlation between intestinal parasites and toxocariasis was detected [P>0.05]. There was also no significant age, gender, and living site difference in toxocara antibody frequency [P>0.05]. Anti toxocara antibody was detected in more than half of children with chronic cough. Intestinal parasites are not associated with hypereosinophilia and show no cross reacting to toxocara antibody