Anti-Toxocara antibodies detected in children attending elementary school in Vitoria , State of Espírito Santo , Brazil : prevalence and associated factors Anticorpos anti-Toxocara em crianças admitidas na escola fundamental em Vitória , Estado do Espírito Santo : prevalência e fatores associados

Introduction: The aim of this study was to evaluate the frequency of anti-Toxocara antibodies in serum from 7-year-old children attending elementary school in Vitória-ES, Brazil and to correlate these antibodies with socio-demographic factors, the presence of intestinal helminths, blood eosinophil numbers, past history of allergy or asthma, and clinical manifestations of helminth infections. Methods: The detection of anti-Toxocara antibodies was performed using an ELISA (Cellabs Pty Ltd)on serum from 391 children who had already been examined by fecal examination and blood cell counts. Data from clinical and physical examinations were obtained for all children. Results: The prevalence of anti-Toxocara antibodies was 51.6%, with no gender differences. No significant differences were observed between positive serology and the presence or absence of intestinal worms (60.3 and 51.7%, respectively; p = 0.286). The only variables significantly related to positive serology were onycophagy and the use of unfiltered water. Although eosinophilia (blood eosinophil count higher than 600/mm3) was significantly related to the presence of a positive ELISA result, this significance disappeared when we considered only children without worms or without a past history of allergy or asthma. No clinical symptoms related to Toxocara infection were observed. Conclusions: There is a high prevalence of anti-Toxocara antibodies in children attending elementary schools in Vitória, which may be partially related to cross-reactivity with intestinal helminths or to a high frequency of infection with a small number of Toxocara eggs.

The prevalence of Toxocara infection as measured by evaluating anti-Toxocara antibodies in serum is variable, with higher prevalence observed in developing countries [1][2][3][4] .Some studies have demonstrated a positive correlation between the presence of anti-Toxocara antibodies and geophagy, onycophagy, a poor hygiene profile and contact with dogs [4][5][6][7] .
A prevalence of 33.3-39% was reported in random samples of children at a Children's Reference Hospital in Vitória 11,18 .In this hospital, a significant association between staphylococcal infection and the presence of anti-Toxocara antibodies was reported 23 .In addition, larva migrans granulomas were observed in 3.2% of livers from routine autopsies at the same hospital 24 .Therefore, Toxocara infection appears to be prevalent in children from Brazil.The Schoolchildren's Health Program of the municipality of Vitoria performs clinical examinations on all children in the first year of public elementary schools.A stool examination, a complete blood cell count and a hemoglobin evaluation are also performed.The aim of the present investigation was to assess the presence of anti-Toxocara antibodies in a representative number of these schoolchildren and to correlate these antibodies with clinical manifestations of infection, socio-economic patterns, hygiene profiles, eosinophil counts and the presence of intestinal helminths.

METHODS RESULTS
Fragoso RP et al -Toxocara infection in schoolchildren Serum samples were collected from 391 schoolchildren in the first year at eight elementary schools randomly selected from 39 schools located in low-income neighborhoods of Vitória.Sociodemographic data including age, gender, family income, parents' schooling, occurrence of geophagy and onycophagy, use of treated water and sewage facilities, and contact with dogs were collected in interviews with parents that agreed to participate in this investigation.
Stool examinations performed using the sedimentation method, blood cells counts and hemoglobin measurements were performed in the Central Laboratory of the municipality of Vitória.The blood cell counts and hemoglobin measurements were performed using automated instruments, with differential counts checked by Giemsastained smears.Aliquots of blood were collected for hematologic studies and were then centrifuged, and the sera were stored at -20ºC until use for anti-Toxocara antibody detection.
Anti-Toxocara antibodies were detected using a commercial test (Toxocara IgG ELISA, Cellabs Pty Ltd, Brookvale, Australia) that uses secretory/excretory antigens of the second stage larvae of Toxocara canis.
The tests were performed according to the manufacturer's instructions.Samples with optical densities (OD) ≤ 0.500 were considered negative, and samples with OD > 0.500 were considered positive.
All children were submitted to physical examination and their history of allergy or other disease was determined during an interview with their parents.
Statistical analysis was performed using SPSS 9.0 for Windows.The frequencies of the variables were calculated with 95% confidence intervals.Following statistical tests, p values < 0.05 were considered significant.

Ethical considerations
The research was approved by the ethics committee of Centro de Ciências da Saúde, Universidade Federal do Espírito Santo.
The detection of anti-Toxocara antibodies was performed on serum from 391 children.Information pertaining to socio-economic status and sanitary conditions was obtained for approximately 340 of these children.All children were 7 years old, the age for admission to elementary schools in Vitória.
The results of the ELISA to measure anti-Toxocara antibodies are shown in Table 2, classified by gender and OD values in the ELISA.The overall prevalence of positive results (OD > 0.500) was 51.6%, without significant gender differences.
Table 3 shows the ELISA results for anti-Toxocara antibodies in children with negative results upon fecal examination, positive results for at least one intestinal nematode and positive or negative results for Ascaris lumbricoides.Although the frequencies of positive antibody tests with different OD were higher in children with Ascaris infection, the differences were not statistically significant.The frequency of positive serology in children without helminth infections was 51.7% (116/224), similar to the overall frequency observed.As the parasite prevalence rates may be underestimated because a single stool sample per child was examined in this study, we also considered the children's past history of worm infection.When considering only children without worms and with no past history of worm infection, the prevalence of positive antibody test results was lower (51/109 or 46.7%) but not significantly different from the overall prevalence observed (46.7 versus 51.7%; p= 0.428).As shown in Table 4, eosinophilia was frequent in the individuals studied (120 of 245 children had an eosinophil count higher than 600cells/mm 3 )and was detected significantly more often in children with positive serology for Toxocara.However, the presence of other intestinal helminths, skin allergy and asthma are important causes of eosinophilia in children.To verify whether a positive test for anti-Toxocara antibodies is a factor that influences peripheral blood eosinophil counts, the proportions of positive and negative antibody results were compared with the frequency of eosinophilia in children with and without intestinal helminths or a past history of skin allergy or asthma.There was no significant difference in eosinophil counts higher than 600cells/mm 3 between the children dogs in household and drinking non-filtered water was significantly associated with positive antibody tests.A multivariate analysis using a logistic regression demonstrated that drinking non-filtered water and onycophagy were independently associated with positive serology for Toxocara.Abnormalities upon physical examination were not found in any children in this study.
A past history of asthma, skin allergy and abdominal pain did not correlate with antibodies against Toxocara.Among 84 children with a past history of asthma and 85 children with a past history of skin allergy, anti-Toxocara antibodies were detected in 46 (54.7%) and 42 (49.4%),frequencies similar to those observed in children without a past history of asthma or skin allergy (86/171, 50.3%;Chi-square test, p=0.475 and 0.825 versus asthma and skin allergy).with positive or negative anti-Toxocara antibody tests.Similarly, the presence of positive antibody test results did not influence the prevalence of eosinophilia in children with intestinal helminths or with a past history of allergy or asthma, thus confirming that the presence of anti-Toxocara antibodies in the serum was not associated with increased blood eosinophils in this sample.
The frequencies of positive anti-Toxocara antibody tests relative to socioeconomic status, sanitary conditions, onycophagy, geophagy and contact with dogs are summarized in Table 5.Low family income, onycophagy, presence of

DISCUSSION
These results demonstrate a high prevalence of anti-Toxocara antibodies in seven-year-old children living in the urban periphery of Vitória.This prevalence was higher than that reported in other Brazilian studies but similar to that observed in Santo Amaro, SP 16 .Even excluding children with intestinal helminths, the frequency of positive serology was still high.Moreover, 110 children had ELISApositive results with OD >1.00, considered by the assay manufacturer to be indicative of more recent infection.
Cross-reactivity against other helminth antigens may be responsible for the high frequency of positive ELISA tests for anti-Toxocara antibodies.This cross-reactivity does exist, mainly against A. lumbricoides 25,26 , and we did not adsorb the serum samples with other nematode antigens before performing the ELISA in this study.However, because the frequency of positive ELISA tests in children without worms was 51.7%, similar to the overall frequency of positive test results, we believe that the high frequency of positive serology observed in this study is not due to cross-reactivity.Moreover, no differences were observed in the frequency of positive results between the individuals with and without intestinal worms or with and without A. lumbricoides(Table 3).This observation confirms that although cross-reactivity exists, the frequency of positive serology in children without intestinal worms is high.Although only one fecal sample per child was examined, our conclusion is strengthened by the observation of an increased percentage of positive antibody tests when we considered both children with parasites and children with a past history of worm infection.The cross-reactivity may be responsible for the frequent detection of ELISA-positive samples with high OD in asymptomatic children.
The high prevalence of anti-Toxocara antibodies in seven-year-old children is consistent with other studies performed in a children's hospital in Vitória.These studies reported prevalences from 30 to 39% of admitted children with positive antibody tests for Toxocara 11,18 , and 3.2% of livers obtained from routine autopsies had granulomas containing Toxocara antigens at the same hospital 24 .
In contrast to other reports 7,11,12,16,21 , we did not observe a significant difference for gender regarding the prevalence of positive tests for anti-Toxocara antibodies.These results are consistent with other studies showing no association between gender and risk of Toxocara infection 22,[27][28][29] .However, positive correlations were observed between positive ELISA tests for Toxocara and low family income, the presence of dogs at home and onycophagia.These results are consistent with the results reported by other authors using samples from different Brazilian cities 7,[13][14][15] .A multivariate analysis demonstrated that onycophagia and the use of unfiltered water were associated with positive antibody tests for Toxocara.An independent association between onycophagia and the presence of anti-Toxocara antibodies was also reported for children in São Paulo 7 .An association between the presence of anti-Toxocara antibodies and the use of unfiltered water has rarely been investigated.However, an investigation in the Amazonia did not reveal a correlation between positive serology for Toxocara and drinking unfiltered water 22 .Some authors have suggested the hypothesis that water contaminated with Toxocara eggs may have a role in Toxocara infection in humans 30 .It is possible that eggs transmitted by neighborhood dust from neighborhoods may contaminate water within the home.In accordance with this possibility, the presence of dog stool in areas near the dwellings was frequently observed during the visits to the children's homes.
Eosinophilia was observed in the samples studied and was more frequent in patientswith positive antibody test results for Toxocara.However, when we evaluated eosinophil counts in children without other causes of eosinophilia, such as intestinal worms, allergy or asthma, this difference disappeared.In accordance with this observation, there was no significant association between positive anti-helminth antibodies and eosinophil counts in children bearing intestinal worms or allergy.Thus, the results presented in this study demonstrate that the presence of anti-Toxocara antibodies in asymptomatic children is not an isolated factor that induces eosinophilia; the increased eosinophil counts in these cases are associated with the presence of other worms and or allergies.

TABLE 4 -Eosinophil counts in seven-year-old children from elementary schools in Vitoria, State of Espírito Santo, Brazil, with or without intestinal helminths or a past history of cutaneous allergy or asthma, according the presence or absence of a positive antibody test for Toxocara canis.
*chi-square test with Yates correction.