Toxocariasis

SUMMARY

In a transversal study on a sample of 386 children and adolescents from an outpatient clinic for filariasis in Recife, Northeast Brazil, the frequency of anti-Toxocara antibodies and its relation to age, gender, number of peripheral cosinophils, Wuchereria bancrofti microfilariae and intestinal helminths was determined. The total anti-Toxocara IgG antibody frequency was 39.4%, by ELISA technique. The difference in frequency between males (40.1%) and females (37.6%) was not statistically significant. The 6 to 10-year-old subset presented the highest frequency of anti-Toxocara antibodies (60%), and within this age group there was a statistically significant male bias. There was also a significant association between the number of cosinophils and the presence of anti-Toxocara antibodies. Intestinal parasite frequency was 52.1%, but no association was found between this data and the presence of anti-Toxocara antibodies. In the present sample, 42.2% of the patients were Wuchereria bancrofti carriers, however, again this was not associated with the presence of anti-Toxocara antibodies. In conclusion, anti-Toxocara antibodies were highly prevalent in this sample. The present data show that there is no cross correlation between anti-Toxocara IgG antibody and the presence of intestinal helminths and filariasis.

KEYWORDS: Toxocariasis; Visceral larva migrans; Anti-Toxocara antibodies; Toxocara canis; Eosinophilia; Wuchereria bancrofti.

INTRODUCTION

RRAVRR et al. (1952) described Visceral Larva Migrans syndrome (VLMS) as a prolonged migration of helminth larvae in the organism of unusual hosts. Toxocara canis is the agent most commonly implicated in the etiology of this illness among human populations'. Infection originates from soil contaminated with parasite eggs eliminated in the feces of common host species, such as dogs and cats. In humans, the parasite completes a somatic-pulmonary biological cycle, which may manifest itself in one of three clinical forms": VLMS (originating from the larval invasion of human tissues); ocular toxocariasis (specific signs and symptoms at the ocular globe); and asymptomatic toxocariasis.

Toxocariasis diagnosis used to be based on clinical data or the occasional Unding of larvae within tissues. An important improvement occurred with the development of Enzyme Linked lmmunosorbcnt Assay (RLISA) using Tmocam canis excretion-secretion antigens that allowed broader epidcmiological studies to be carried out. It has been found that the parasitic infection prevalence varies widely in the globe and even within a single country, due to its epidcmiological characteristics4,14,18,21,24,25,31.

In many Brazilian regions, particularly in the Northeast countryside, one should expect to find a higher prevalence than those presently reported in the U.S. or Europe. Despite the favorable cpidcmiological conditions lor mass occurrence of human infection, such as the large number of 71 cam'i infected street dogs^, there is a surprisingly low frequency ofVLMS cases diagnosed in the Brazilian Northeast. However, few epidcmiological studies have been carried out on this subject, thus preventing an accurate estimation of the prevalence of toxocariasis for the region. Consequently, these conditions may signify one of the largest unrecognized parasitic infections in Recife and in view of the potential morbidity further investigation is warranted.

The present study was done in order to determine the frequency of anti-Tcurocam antibodies in the serum of children and adolescents exposed to other endemic parasitosis in Recife, as well as to verify the possible role of these infections on the anti-Toxocam scrology.

MATERIAL AND METHODS

A transversal study was carried out on children and adolescents attended at Centre de Pcsquisas Aggcu Magalhacs (CPqAM/Piocruz) outpatient clinic, a national reference center for lymphatic filariasis, in Recife city (Brazilian Northeast) from 1987 to 1996.

Tlic sample studied consisted of 5(X) individuals, nil less than 19 years old. Taking into account an 11 %^' expected prevalence and 9?% confidence interval, a minimum of 326 individuals was estimated. In fact, a total ol" 386 patients were selected and examined, of which 269 were male (69.7%) and 117 (30.3%) were female. Individuals without scrological samples available were excluded.

The following routine laboratory exams were performed: sera antiTb.vocom antibodies; cosinophils absolute count and tests for intestinal parasites and Wuchereria bancrrfti microfilariac in the blood sample.

Bosinophils absolute count was obtained by direct inspection in a Neubauer chamber-', with a reference value? of 500 cosinophils/mm^sup 3^ Hor larvae, eggs and cysts study of the intestinal parasites, the Lutz method as well as the Baerman-Moracs method were applied, using one or more stool samples for diagnosis. Protozoan species were not identified.

Membrane nitration technique was used in filariasis study, as described by DKNNIS & KHAN11. Venous blood samples were collected between 11:00 p.m. and 1:00 a.m., according to the circadian periodicity of microfilariac11.

Total IgG anti-Tb.Yocom antibodies study was carried out by KLISA immuno-cny.ymatic method, using secretory and excretory antigens from second stage larvae (L^sub 2^) cultivated m ivVro8.9. All sera were adsorbed with total Ascaris antigcnic extract to avoid cross reactions between yovocura antigens and Ascaris antibodies1,22. The cutoff density point in serological assays varied from 0.520 to 0.720 and was determined for every test using the mean of the negative controls plus two standard deviations (2 SD).

KPi Info 6.04 software was used for statistical analysis. A descriptive univariatc analysis was carried out to verify the frequency of the variables and the scroprevalcncc of anti-Tb.wcm antibodies in the total sample, pooled according to gender and age. The relative proportions were calculated with a confidence interval of 95%. To identify possible associations between dependent variable (scropositivity for Tb.tocam) and independent variables Pcnrson chi-squarc test with Yates correction was used. The significance level was set at 5%.

The protocol design as well as the use of biological fluid samples stocked at our Institution had the approval of the local ethical committee.

RESULTS

Genem characterizationof population study: Eosinophil count was carried out on 303 (78.5%) of the 386 individuals enrolled. Of these, 67.7% were cosinophilic (values above 500 cosinophils/rhm^sup 3^), whereas 35.9% presented hypcrcosinophilia, i.e. values above 1000 cosinophils/ mm^sup 3^

Intestinal helminths were present in 52.1% of individuals that underwent coprological examination. Table I describes the intestinal parasites found and summarizes the frequency, as well as other characteristics of the sample population.

Wuchereria bancrfti microfilariaea(mf) study was carried out on 353 individuals (91.4% of overall sample), and 42.2% were diagnosed as carriers. The mean density was 410 mf/ml (range 1 to 12.650 mf/ml).

Frequency of anli-Toxocara antibodies in relation to gender, age, llie presence of intestinal parasites or W. bancrofti microfilariae: Anti-Toxocara antibodies frequency was 39.4% (152/386). The antibody frequency among male individuals was 40.1 % ( 108/269) while in females it was 37.6% (44/1 17). This difference was not statistically significant (Table 2). However, when a stratified analysis by age group was carried out, a 88.9% antibodies frequency was found in males aged 6 to 10, compared to 40.7% in age paired females. Thus, there was a statistically significant greater risk (2.1 times) of being seropositive for males in this age group.

The highest seropositivity frequency was found in the 6 to 10-year-old subgroup, representing 60% (27/45) of positive individuals. This was followed by individuals aged 11 to 15 years; less than or equal to 5 years; and 16 to 18 years with 46.4% (51/110), 32.1% (9/28) and 32% (65/203) seropositivity, respectively. all of these subgroups presented statistically significant differences (Table 2).

Considering only those individuals with positive scrology that also underwent studies IOr Wiuchereai 52.8% (75/142) were not carriers while 47.2% (67/142) were carriers. This difference was not statistically significant.

Amund 51.7% of seropositive children and adolescents presented some type of intestinal parasite whereas no parasite was detected in 48.3%. This difference was not statistically significant.

In relation to seropositive individuals with an eosinophil count, 12% (14/117) presented normal eosinophil values, whereas 53.8% (63/117) had hypereosinophilia. Individuals with higher eosinophil counts (> 3000 cells/mm^sup 3^) also presented a greater frequency of anti-Tkrocam antibodies (61.3%) (Pig. I). Only 35 individuals had hypereosinophilia without associated intestinal parasitosis or filariasis and among these, 16 presented anti-7b.iocam antibodies.

The relation between the number of cosinophils and seropositivity frequencies was statistically significant, with a 4.29 times greater seropositivity risk in individuals with values above 3000 cosinophils/mm^sup 3^ when compared to those with less than or equal to 500 eosinophils/mn^sup 3^ (Pig. I).

DISCUSSION

We are aware that the anti-Tb.tocam antibodies frequency in the current study may not reflect its real prevalence in the population of the Recife metropolitan region. Nevertheless, the present data arc relevant as can figure out the Toxocara prevalence in a population sample exposed to other intestinal parasitosis, especially Wuchereria bancrofti.

Our Institution's outpatient clinic attends a large number of individuals with peripheral cosinophilia. This eventual laboratory finding is the major reason for referral to our ambulatory and leads to a higher overall hypereosinophilic patient profile in relation to the general population. Since the presence of peripheral eosinophilia is one of the laboratorial criteria considered for a presumed Toxocara infection, it should he stated that the present sample population had a greater chance of presenting imu-Toxocara antibodies.

The high frequency of anti-Toxocara antibodies found in this study was similar to reports in Brazil23,26,33 and other countries17,21 but nevertheless higher than that described by some authors4,14,24,25. However, the comparison between the present results and these reports is difficult due to several factors, such as: scroepidemiological studies based on a small number of children; heterogeneity of individuals studied (i.e. blood donors, hospitalized patients or high risk subgroups for toxocariasis); non-standardized laboratory procedures; and different cutoff points in the various serodiagnostic techniques.

Intestinal parasite infection was detected in a high percentage of individuals, resulting in a significant frequency of gcohclminths between the detected parasites. They are well known agents for the onset of peripheral eosinophilia28. The high frequency of intestinal helminths observed in the present study suggests that other risk factors described for Toxocara infection14,21,31, such as poor hygiene habits and inadequate sanitation, must have contributed14,21,31.

The frequency analysis of anti-Toxocara antibodies in a population survey from a tropical area with serious sanitary and socioeconomic deficiency is further complicated by potential cross reactivity between a large variety of other parasites to which a given individual may have been exposed. This could lead to false-positive results21.

Recife city is considered to be an endemic area of bancroftian filariasis34.Thus, the microfilariae examination of serum samples could be important to determine whether a cross reactivity of Toxocara with other nematodes is present in individuals with positive scrology. However, present data discarded a correlation between the presence of W. bancrofti microfilariac and anti-Toxocara antibodies.

No significant difference was observed between the frequency of intestinal parasites and anti-Toxocara antibodies. Thus, one should be aware of the possibility that these infections overlap in the population.

We found a significant gender difference in antibody frequency only in 6 to 10-year-old subgroup, with a male bias. Our data corroborates other reports that indicate greater frequency of Tarocara infection among males in certain age groups9,12,16,19,29. Taking into account the Brazilian culture, this difference may be due to more restricted outdoor activities for female children.

The peak antibody frequency found in the present study (6 to 10-year-old subgroup) was similar to that described by CHIEFFI et al. in a population study in Santos city, Sao Paulo State, in southeastern Brazil6. They reported a 13.1% scroprevalcnce in individuals less than 15 years of age, hut the prevalence among those in the 5 to 10-year-old group reached 19.0%.

A high frequency of anti-Toxocara antibodies among pediatric patients seems to be unquestionable9,16,35, but there has been a growing number of reports indicating the presence of infection in adults14,30. This is especially relevant to regions considered to be endemic areas for toxocariasis (even for adults), such as the southeast of France15. Therefore, while 2 to 5-year-old children have always been considered the classic age group for susceptibility to VLMS, one should not make the error of considering that toxocariasis is an exclusively childhood illness.

BEAVER2 reported a close relation between eosinophilia and visceral larva migrans. Prior to the development of ELISA technique, this criterion for cosinophilia (with mainly extreme values) was adopted as an important laboratorial indicator of toxocariasis. Even today, it is considered a classic criterion of visceral larva migrans. However, the absence of an elevated number of peripheral eosinophils docs not exclude the possibility of toxocariasis30.

The literature also indicates that cosinophilia in Toxocara seropositive individuals seems to be age related, as it is more common in children than in adults32.

Analyzing the present data, we were able to identify other probable causes for cosinophilia besides a possible Thxocam infection, such as the presence of intestinal parasites and filarial infection. The latter appears to be particularly probable in patients with the clinical form of Tropical Pulmonary Eosinophilia (TPE), with elevated values of peripheral cosinophils.

The initial objective of this work was to use anti-Tbjrocam antibody ELlSA to asses the toxocariasis infection at an outpatient clinic in Recife, a recognized endemic area of filariasis and geohelminthiasis. Although the present data docs not allow a more profound analysis of all risk factors for Toxocam infection, it strongly suggests that the Brazilian Northeast has a high human toxocariasis prevalence, which has so far been underestimated and, consequently, under-diagnosed. On the other hand, in the present population sample the presence of filariasis apparently docs not alter the ELlSA test with secretion and excretion L2 antigen, which strengthens the relevance of this nosology within the region.

RESUMO

Toxocanasc humaiia: frcqucncia dc anticorpos anti-Toxocara em criancas c adolescentes atcndidos cm um ambulatorio cspccializado dc filariosc linfntica em Recife, Nordcstc do Brasil

Atravis dc cstudo do tipo transversal com amostra constitufda por 386 criancas c adolescentes atendidos em urn ambulatorio especializado dc filnriosc, do Recife, nordeste do Brasil, determinou-se a frequencia de anticorpos anti-Taxocom e sua relacao com faixa etaria, sexo, numero de cosinofilos perifiricos, microfilarias de WifcAergn'a bancrofti e parasites intestinais. A frequencia encontrada de anticorpos IgG total anti-Thxocam, rcalizada atravcs da tecnica de ELISA, foi de 39,4%, com 40,1% no sexo masculino c 37,6% no fcminino, difcrcnca esta scm significancia estatfstica. O grupo com maior frcqucncia dc anticorpos anti-Toxocara foi o de 6-10 anos (60%) e, apenas nessa faixa etaria, cncontrou-se uma diferenca cstatisticamente signi ficante quanto ao sexo, com predomfnio do masculino. Observou-se associacao estatisticamentc signi ficantc entre o numero de cosinofilos c a presenca dc anticorpos anti-Toxocara. A frequcncia de parasites intestinais foi de 52,1 %, porom sem associacao entre este achado e a prescnca de anticorpos anti-Toxocara. Na presente analise, 42,2% dos pacienlcs cram porladores de microfilarias de Wuchereria bancrofli, porcm csla infeccao nao estcve associada a prcsenca de anticorpos anti-Toxocara o que sugere que nao houvc cruzamento do ELlSA com a presence de parasitoses intcslinais e filariose.

ACKNOWLEDGEMENTS

The authors are indebted to Prof. Andre Frei re Futlado and Dr. Luiz Bezera de Carvalho for their valuable suggestions.

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Received: 1 August 2003

Accepted: 24 March 2004

Ana Maria AGUlAR-SAISTOS(I), Luiz D. ANDRADE(I), Zulma MEDEIROS(2), Pcdro Paulo CHIEFKI(S), Suzana /,. LESCANO(S) & Emilia P. PERKZ(I)

(1) Centre de Pcsquisas Aggeu Magalhaes/FIOCRUZ, Av. Moracs do Kcgo s/n, Campus da Univcrsidadc Federal de Pcrnambuco, Cidade Universitâria, 50670-420 Recife, PE, Brasil. Phone: (81) 33012573; fax: (8l) 3453 2449. E-mail: amas@cpqam.fiocruz.br

(2) Centra de Pesquisas Aggcii Magalhaes/FIOCRUZ, Institute de Ciencias Biologicas/UPE.

(3) Instituto de Medicina Tropical de Sao Paulo/USP, Av. Dr. Eneas de Carvalho Aguiar 470, 05403-000 S. Paulo, SP, Brasil.

Correspondence to: Ana Maria AGUIAR-SANTOS, Av. Moraes do Rego s/n, Campus da Universidade Federal de Pernambuco, Cidade Universitaria, 50670-420 Recife, PE, Brazil. E-mail: ailias@cpqai.fiocruz.br

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