Sensitivity and specificity of recombinant proteins in Toxocara spp. for serodiagnosis in humans: Differences in adult and child populations.

Toxocariasis is a neglected zoonosis that affects children and adults. Recombinant proteins have been widely investigated for diagnosis, achieving high sensitivity and specificity in an overall population; however, little is known about age as a factor in its application. This study aims to investigate the diagnostic potential of Toxocara canis TES-30 and TES-120 recombinant proteins in humans, differentiating between its performance in children and adults. Serum samples collected from children and adults seropositive to Toxocara spp. were tested with indirect ELISA using T. canis TES-30 and TES-120 recombinant proteins produced in Escherichia coli. While rTES-30 sensitivity was not affected by age (81.8% in children and 87% in adults), rTES-120 sensitivity severely decreased in children to only 63.6%, down from 95.7% in adults. Furthermore, the sensitivity of rTES-30 increased to 97.8% after Western blotting confirmation. High specificity (>94%) against other geohelminths was reported for both recombinant proteins. Our study favors the use of rTES-30 with total IgG as the primary antibody in an indirect ELISA assay as a tool for epidemiological human studies.

Toxocara spp. seroprevalence in pregnant women in Brasília, Brazil.

INTRODUCTION:: The impact of gestational toxocariasis is an understudied topic on female reproductive health. We estimated anti-Toxocara IgG prevalence among pregnant women in Brasília, Brazil, and investigated the association of the infection with history of abortion and contact with pets. METHODS:: Infection was diagnosed using ELISA with excretory/secretory antigens. Participant information was obtained via questionnaires. RESULTS:: Of 311 pregnant women, 23 were anti-Toxocara IgG positive. Twenty-two percent of anti-Toxocara IgG-positive participants and 26% had previously miscarried. Previous contact with pets was associated with higher toxocariasis prevalence. CONCLUSIONS:: A direct relationship between toxocariasis and contact with pets was observed, but there was no relationship with the miscarriage prevalence.

Evaluation of Toxocara canis Glycosylated TES Produced in Pichia pastoris for Immunodiagnosis of Human Toxocariasis

Abstract Recombinant proteins are a suggested alternative for the diagnosis of toxocariasis. The current Escherichia coli recombinant protein overexpression system usually produces insoluble products. As an alternative, yeast such as Pichia pastoris have secretory mechanisms, which could diminish the cost and time for production. This study aimed to produce recombinant proteins in Pichia pastoris and verify their sensibility and specificity in an indirect ELISA assay. Two sequences (rTES-30 and rTES-120) of Toxocara canis excretory-secretory antigens were cloned in a pPICZαB vector and expressed in P. pastoris KM71H. Sera samples collected from human adults infected by Toxocara spp. were tested by indirect ELISA using rTES-30 and rTES-120 as antigens. Recombinant proteins were detected at 72 hours after induction, in the supernatant, as pure bands between 60~70 kDa with hyperglycosylation. Regarding diagnosis potential, recombinant antigens had high specificity (95.6%); however, sensitivity was 55.6% for rTES-30 and 68.9% for rTES-120. Further deglycosylation of the P. pastoris antigens did not seem to affect ELISA performance (p>0.05). The low sensitivity in the serodiagnosis diminished any advantage that P. pastoris expression could have. Therefore, we do not recommend P. pastoris recombinant TES production as an alternative for the diagnosis of toxocariasis.

[Frequency of seropositivity to Toxocara canis in children of different socioeconomic strata]. / Freqüência de soropositividade para antígenos de Toxocara canis em crianças de classes sociais diferentes.

Frequency of seropositivity for Toxocara in children from different socioeconomic strata in the city of Brasilia (Brazil) was measured. Six hundred and two children of both sexes, aged one to 12 years were distributed in two socioeconomically distinct groups. The samples of sera of the first group were obtained from blood drawn for routine tests in the laboratory of a public hospital attending children from low-income families. Samples from the second group were obtained from private laboratories attending children from middle-class families. Anti-toxocara antibodies were detected by ELISA, using Toxocara canis excretory-secretory antigens previously absorbed with Ascaris suum extract. The prevalence of seropositivity was 21.8% (66/302) in the first group and 3% (9/300) in the second (p< 0.0001). No differences in frequency according to age or sex could be detected. Our results suggest a high prevalence of childhood toxocariasis in Brasilia, with children from lower income brackets being the most affected.

An alternate technique for isolation of Toxocara canis excretory-secretory antigens / Uma técnica alternativa para o isolamento de Toxocara canis excreção e secreção de antígenos

The aim of the present study was to test the effectiveness of a sausage-casing membrane for dialysis of Toxocara excretory-secretory antigens (TES). The protein concentrated by the tested membrane was compared with that obtained using a Sigma commercial membrane, as were the protein fractions found by polyacrylamide gel electrophoresis. Standard positive and negative serum samples were evaluated in an ELISA immunoassay, and equivalent data were obtained in all steps, indicating that the sausage-casing membrane is efficient, besides being less expensive to process.

O objetivo do presente estudo foi testar a eficácia de uma membrana utilizada para o preparo de embutidos, na obtenção do antígeno de excreção e secreção de Toxocara (TES). A concentração protéica foi comparada com a obtida com a membrana Sigma tanto quanto as frações protéicas separadas por eletroforese em gel de poliacrilamida. Amostras de soros padrão positivo e negativo foram avaliadas no teste imunoenzimático ELISA. Dados equivalentes foram observados em todas as etapas, sugerindo que a membrana possa ser utilizada para diálise por ser eficiente e de menor custo no preparo do antígeno.

A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay.

A serological follow-up study was carried out on 27 children (1-12 years old) with visceral and/or ocular toxocariasis, after treatment with thiabendazole. A total of 159 serum samples were collected in a period ranging from 22-116 months. Enzyme-linked immunosorbent assays (IgG, IgA, and IgE ELISA) were standardized, using excretory-secretory antigens obtained from the second-stage larvae of a Toxocara canis culture. The sensitivity found for the IgG, IgA, and IgE ELISA, as determined in visceral toxocariasis patients, was 100%, 47.8%, and 78.3%, respectively. Approximately 84% of the patients presented single or multiple parasitosis, as diagnosed by stool examination, yet such variables did not appear to affect the anti-Toxocara immune response. Titers of specific IgE antibody showed a significant decrease during the first year after treatment, followed by a decrease in the IgA titers in the second year, and in the IgG titers from the fourth year onwards. Sera from all patients presented high avidity IgG antibodies, indicating that they were in the chronic phase of the disease. Moreover, 1 year after treatment, the level of leukocytes, eosinophils, and anti-A isohemagglutinin in patients decreased significantly. The present data suggest that IgE antibodies plus eosinophil counts are helpful parameters for patient follow-up after chemotherapy.

Freqüência de soropositividade para antígenos de Toxocara canis em crianças de classes sociais diferentes / Frequency of seropositivity to Toxocara canis in children of different socioeconomic strata

Para estudar a freqüência da infecçäo pelo Toxocara canis em crianças de classes sociais diferentes de Brasília, Brasil, foram testados soros de 602 crianças de ambos os sexos, de 1 a 12 anos, distribuídas em dois grupos representativos de condições socioeconômicas distintas. As amostras do primeiro grupo foram obtidas em laboratório público que atende bairros pobres. As do segundo grupo foram colhidas em laboratório privado, que serve à classe média. Os anticorpos anti-Toxocara foram detectados pelo método ELISA, com antígenos de Toxocara canis, e absorçäo com antígenos do Ascaris suum. A prevalência de soropositividade foi de 21,8 por cento (66/302) no primeiro grupo e de 3 por cento (9/300) no segundo (p<0,0001). Näo houve diferença quanto ao gênero ou idade das crianças. Os resultados sugerem significativa prevalência de toxocaríase na populaçäo infantil de Brasília, com predomínio nas classes sociais menos favorecidas economicamente