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    Geographic location of the study area. Jaboatão dos Guararapes is situated in the suburbs of Recife, the capital of the State of Pernambuco. Administratively, the area belongs to Região Metropolitana do Recife.

  • View in gallery

    Specificity of the recombinant Toxocara canis antigen. Thirteen children who expelled only Ascaris lumbricoides eggs showed positive reactions against homologous ascarid antigen (A), but none of them reacted with the recombinant T. canis antigen (B). Closed circles indicate serum samples from the 13 children. Each symbols in the panel A corresponds to those in panel B except for the positive controls. Triangles and open circles denote positive and negative controls, respectively. The cut-off value is based on three times the optical density at 415 nm (OD415) value for a pooled negative serum sample.

  • View in gallery

    Enzyme-linked immunosorbent assay results using recombinant Toxocara canis antigen. Serum samples examined were divided into six groups based on the absence or presence of helminth eggs. A, No helminth eggs; B, Only Ascaris lumbricoides eggs; C, Only Trichuris trichiura eggs; D, Either hookworm (top two circles), Schistosoma mansoni (bottom and third circle from the bottom), Strongyloides stercoralis (fourth circle from the bottom), or Enterobius vermicularis (second circle from the bottom) eggs; E, Mixed helminth eggs such as A. lumbricoides, T. trichiura, hookworm, and/or Hymenolepis nana; F, individuals without information; G, positive (triangle) and negative controls (open circle) for toxocariasis. The broken line indicates the cut-off value. OD415 = optical density at 415 nm.

  • View in gallery

    Dot-blot assay using selected serum samples. In positive cases, the spots appear in the right side upper square. Spots in the left side lower grids are positive controls for secondary antibody. The two panels at the top row (A1 and A2) indicate negative and positive controls for the enzyme-linked immunosorbent assay (ELISA)-positive group. Results from A3 to C8 correspond to code numbers 401–10306 as in Table 1. Panels D1 and D2 are negative and positive controls for ELISA-negative group, and results from D3 to F9 correspond to code numbers 101–10104 in order.

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PREVALENCE OF TOXOCARIASIS IN NORTHEASTERN BRAZIL BASED ON SEROLOGY USING RECOMBINANT TOXOCARA CANIS ANTIGEN

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  • 1 Departamento de Bioquímica, Laboratório de Imunopatología Keizo Asami, Universidade Federal de Pernambuco, e Departamento de Parasitología, Centro de Pesquisas Aggeu Magalhães-FIOCRUZ, Recife, Pernambuco, Brazil; Section of Environmental Parasitology, Tokyo Medical and Dental University Graduate School, Tokyo. Japan; Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo, Japan; Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Tokyo, Japan; Department of Parasitology, Asahikawa Medical College, Asahikawa, Japan

To evaluate the prevalence of toxocariasis in children in Jaboatão dos Guararapes, Pernambuco in northeastern Brazil, 215 serum samples were examined by an enzyme-linked immunosorbent assay (ELISA) using a recombinant Toxocara canis antigen. In the ELISA, 26 (12.1%) of 215 subjects were positive. In a dot-blot assay using 53 of 215 serum samples, the diagnostic results correlated with those obtained by the ELISA. Moreover, it has been confirmed that the recombinant T. canis antigen was highly specific for toxocariasis by ELISA using serum samples positive for antibody to Ascaris lumbricoides. Considering the specificity of the recombinant antigen to toxocariasis, the ELISA or dot-blot assay using the recombinant T. canis antigen is recommended in tropical and sub-tropical regions where various parasitic infections are commonly endemic.

INTRODUCTION

Toxocariasis is an important zoonosis caused by the infection of larvae with the dog nematode Toxocara canis or the cat nematode Toxocara cati.1 In humans, when Toxocara eggs containing infective larvae are accidentally ingested, the larvae hatch in the small intestine and migrate through somatic organs, preferably the liver and eyes. Clinically, there are two types of toxocariasis: visceral larva migrans and ocular larva migrans. Visceral larva migrans is characterized by chronic weakness, abdominal pain, diverse signs of allergy, or hypereosinophilia.2,3 Ocular larva migrans occurs when larvae become trapped in the eyes, leading to uveitis and optic papillitis.4 Toxocariasis has also been proposed as a potential etiology in neurologic disorders when the larvae migrate in the central nervous system.5–8

In the immunodiagnosis of toxocariasis in humans, excretory-secretory (ES) antigens have been widely used.7,9–14 However, it has been shown that ES antigens are cross-reactive against serum samples from patients with a variety of helminth infections.7,12,15–18 Therefore, to overcome such problems, a recombinant T. canis antigen has been developed18 and its high specificity and usefulness have been demonstrated.19,20

In Brazil, seroepidemiologic studies on human toxocariasis have been carried out in Recife, Pernambuco,21,22 Butantã, São Paulo,23,24 Vitória, Espírito Santo,25 Campinas, São Paulo,26 and Brasilia, Distrito Federal,27 and it has been reported that the prevalence rates of toxocariasis reached approximately 40% in some studies.21–23 Here we report the prevalence rate of toxocariasis based on serologic analysis using the recombinant T. canis antigen in children in the suburb of Recife in northeastern Brazil. In addition, the specificity of recombinant T. canis antigen was also evaluated using serum samples from individuals infected with Ascaris lumbricoides, which occurs commonly in subtropical and tropical areas.

MATERIALS AND METHODS

Study area.

The study area, Jaboatão dos Guararapes, is situated at approximately 20 km southwest of Recife, the capital of the State of Pernambuco, Brazil (Figure 1). The area is rural with approximately 580,000 inhabitants and local residents who make a living cultivating sugarcane and raising livestock. Socioeconomic standings are low and sanitary facilities are poor. It has also been reported that schistosomiasis mansoni is still endemic in the surrounding district.28,29

Human serum samples.

Serum samples were collected from 215 children (age range = 1–17 years), after informed consent was obtained, for epidemiologic and clinical surveys on helminth infections. Personal information on sex and stool examination was available for 198 of the 215 children. This group was composed of 104 males and 94 females. Based on stool examinations, eggs of A. lumbricoides, Trichuris trichiura, Strongyloides stercoralis, hookworms, Schistosoma mansoni, Enterobius vermicularis, and/or Hymenolepis nana were detected from 163 of 198 individuals (82%). Physical examinations were also performed. This study was reviewed and approved by the Ethical Committee of the Centro de Ciências da Saúde of the Universidade Federal de Pernambuco, Brazil.

Preparation of somatic antigens from Ascaris suum adult worms.

To confirm the specificity of recombinant T. canis antigen, serum samples reactive against somatic antigens from adult worms of A. suum were screened. The antigen was prepared as follows. Ascarid worms stored at −80°C were homogenized in chilled 0.15 M phosphate-buffered saline (PBS, pH 7.2) and centrifuged at 10,000 rpm for 10 minutes at 4°C. The resulting supernatant was used as a somatic antigen. The protein concentration was estimated using the bicinchoninic protein assay kit (Pierce, Rockford, IL) and adjusted to a concentration of 5 μg/mL for the enzyme-linked immunosorbent assay (ELISA).

Preparation of a recombinant T. canis antigen.

The recombinant T. canis larval antigen was prepared as previously described.18 Briefly, insoluble antigen produced in bacteria was solubilized in 8 M urea and purified using a TALON metal affinity resin column (Clontech, Palo Alto, CA). The purified antigen was stored at −80°C until use. Protein content was estimated as described earlier in this report.

Enzyme-linked immunosorbent assay and dot-blot assay.

The ELISA using the recombinant T. canis antigen was performed as previously described, except for the use of 3,3,5,5-tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) as the substrate.20 Briefly, 96-well microtiter plates were coated with the recombinant antigen at a concentration of 125 ng/mL (100 μL/well) for two hours at 37°C, washed three times with PBS containing 0.05% Tween 20 (PBS-T), and then probed with 100 μL of human serum sample (1:200 dilution with PBS-T containing 1% bovine serum albumin [PBS-T/BSA]) for 40 minutes at 37°C. After washing with PBS-T, 100 μL of horseradish peroxidase conjugated with rabbit anti-human IgG (Cappel, Aurora, OH) were added into each well and incubated for 35 minutes at 37°C. For color development, 50 μL of TMB were added into each well and incubated for five minutes at room temperature. The reaction was terminated by adding 25 μL of 2 M H2SO4. Optical density (OD) at 415 nm was monitored with a Microplate Reader (Bio-Rad Laboratories, Hercules, CA). The cut-off point was set based on three times the OD value for a negative serum sample pooled from 40 Japanese individuals who were parasite free.

For the dot-blot assay, 53 serum samples were selected based on the results of the ELISA; 26 were ELISA-positive samples and 27 were ELISA-negative samples from individuals infected with helminths and those not infected (Table 1). Nitrocellulose membranes with a 3 × 3 mm grid (pore size = 0.45 μm; Toyo Roshi Ltd., Tokyo, Japan) were used. The recombinant T. canis antigen (100 ng/0.5 μL) diluted with PBS was spotted onto the right side upper grids. As a positive control for the second antibody, 0.5 μL (1:200 dilution) of a serum sample pooled from 40 uninfected Japanese individuals were spotted onto the left side lower grids. The membranes were then air-dried, soaked in PBS-T/BSA for 30 minutes at room temperature, aspirated to remove the excess blocking solution, and stored at 4°C until use. In the assay, 50 μL of human serum diluted 1:200 with PBS-T/BSA were placed onto the membrane and probed for 40 min at 37°C in the humid box. After washing three times with PBS-T, the membranes were incubated with 50 μL of rabbit anti-human IgG conjugated with horseradish peroxidase (1:750 dilution) at 37°C for 40 minutes. For color development, the membranes were incubated with a substrate solution (0.4% 4-chloro-1-naphthol containing 0.005% H2O2 in 46% ethanol) for 5–10 minutes. The color reaction was terminated in an excess of distilled water. Positive results were visualized by a well-defined purplish color on the membranes.

RESULTS

Although the specificity of the recombinant T. canis antigen has been assessed in a previous study,20 it has also been further evaluated in this study. For this purpose, serum samples from 42 individuals who excreted only A. lumbricoides eggs were tested by the ELISA using A. suum antigen and 13 samples were positive (Panel A, Figure 2). We evaluated the reactivity of these serum samples against the recombinant T. canis antigen. None of these samples reacted with the recombinant antigen, indicating that the recombinant T. canis antigen is highly specific (Panel B, Figure 2). Figure 3 shows the ELISA results against the recombinant T. canis antigen of 215 serum samples. All samples examined were divided into six groups based on the absence and presence of helminth infections: 36 from individuals without any helminth eggs (panel A), 42 from individuals with A. lumbricoides eggs only (panel B), 26 from individuals with T. trichiura eggs only (panel C), 6 from individuals with either hookworm, S. mansoni, S. stercoralis, or E. vermicularis (panel D), 88 from individuals with mixed helminth eggs (panel E), and 17 from individuals without information (panel F). Panel G shows positive and negative controls. Twenty-six cases were positive in the ELISA. Based on the clinical cases that we have experienced, we believe that cases with higher OD values are strongly suspected to be visceral toxocariasis. Other abnormalities, such as hepatomegaly or eosinophilia, were not found in positive cases (Table 1).

Based on the ELISA results, the usefulness of the dot-blot assay was evaluated using 53 serum samples (Figure 4). The three rows (A3 to C8) show results from the ELISA-positive group. These samples correspond to the code numbers in Table 1 in order. In positive samples, spots appeared in the right side upper grid in all samples, although very faint spots appeared in B5 (code no. 7802 in Table 1), C4 (code no. 9602), and C5 (code no. 9603). In contrast, no spot appeared in 27 ELISA-negative serum samples (D3 to F9). Spots did not appear in the negative controls (A1 and D1) and clear spots appeared in the positive controls (A2 and D2).

DISCUSSION

In the present study, the prevalence rate of toxocariasis in Jaboatão dos Guararapes in northeastern Brazil was examined by ELISA and dot-blot assay using recombinant T. canis antigen. In previous studies on human toxocariasis in Brazil, it has been reported that prevalence rates reached approximately 40% in different study areas.21–24,27 However, when compared with results obtained in the present study, this seems to be extremely high. The high prevalence rates could be due to the cross-reactivity of antigens used. Indeed, native T. canis ES antigens have been shown to cross-reacted with various helminth infections.7,12,16,17,20,30 It has also reported that the prevalence rates are affected by the socioeconomic strata of the population examined.24,27

Serologic tests based on an ELISA showed that 26 (12.1%) of 215 subjects showed positive reactions against the recombinant T. canis antigen and at least 19 of 26 individuals were infected with A. lumbricoides, T. trichiura, hookworms, and other parasites (Table 1). In cases of A. lumbricoides infection, the possibility that transitional antibody responses due to the migration of the ascarid nematode larvae cannot be ruled out. However, no cross-reaction was observed in an ELISA using serum samples from mice experimentally infected with A. suum.20 Since serum samples positive against T. trichiura and hookworm antigens were not available in the present study, the recombinant T. canis antigen does not seem to cross-react with serum samples from individuals infected with these nematodes.20 Considering high specificity of the recombinant T. canis antigen and the lifestyles of the local people that are closely associated with dogs, we believe that the positive cases are mixed infections with T. canis rather than cross-reactions.

The ELISA is a simple method to perform, but expensive equipment such as the microplate reader is required. In contrast to the ELISA, the dot-blot assay is a simpler and convenient procedure. It does not require any expensive facilities, the membranes preadsorbed with antigens can be stored at 4°C for many years, and the membranes are stable at room temperature and can be shipped if requested. In the present study, it has been demonstrated that the dot-blot assay using recombinant T. canis antigen provides highly reliable diagnostic results for the serodiagnosis of human toxocariasis. In particular, the technique is very useful in many subtropical or tropical areas where laboratory facilities are limited and various parasitic diseases commonly occur. Most recently, a new diagnostic format has been developed using polysiloxane/ polyvinyl alcohol beads coupled with recombinant T. canis antigen for the immunodiagnosis of human toxocariasis and its usefulness has been demonstrated.31 Thus, the use of the recombinant T. canis antigen, even in different diagnostic methods, would be recommended not only for routine diagnosis, but also for seroepidemiologic surveys of toxocariasis in humans. In addition, considering the prevalence rate and clinical symptoms of toxocariasis, more attention should be paid to ocular or visceral toxocariasis. A clinical survey based on serologic results has begun in the study area.

Table 1

Diagnostic results of selected serum samples from children*

Code no.Age (years)/sexELISADot-blot assayStool examinationsClinical findings†
* ELISA = enzyme-linked immunosorbent assay; A. = Ascaris; T. = Trichura; NA = not available; S. = Strongyloides; E. = Enterobius; NC = negative pooled serum; PC = positive serum from a toxocariasis patient.
† Hepatomegaly and splenomegaly were not observed.
4014/F0.143+A. lumbricoides and T. trichiura eggs (+)Abdominal pain, diarrhea (sometimes)
250512/M0.227+A. lumbricoides and T. trichiura eggs (+)None
32023/F0.145+No helminth eggsVomiting
33025/M0.167+T. trichiura eggs (+)Abdominal pain, diarrhea (sometimes)
37013/F0.2+T. trichiura eggs (+)Abdominal pain
37026/M0.239+A. lumbricoides eggs (+)Abdominal pain, diarrhea
420410/M0.171+A. lumbricoides eggs (+)None
420815/F0.185+Hookworm eggs (+)None
4802NA0.154+No informationNone
4804NA0.537+No informationNone
700410/F0.158+A. lumbricoides eggs (+)Constipation
78018/F0.25+A. lumbricoides eggs (+)Constipation
780211/F0.171+No helminth eggsAbdominal pain
81023/M0.205+T. trichiura eggs (+)Abdominal pain, diarrhea (sometimes)
89013/M0.213+Hookworm eggs (+)Abdominal pain
89039/F0.4+Hookworm and T. trichiura eggs (+)Abdominal pain
890411/F0.212+A. lumbricoides, hookworm, and T. trichiura eggs (+)Abdominal pain
890513/F0.246+A. lumbricoides, hookworm, and T. trichiura eggs (+)Abdominal pain
890617/M0.178+A. lumbricoides, hookworm, and T. trichiura eggs (+)Abdominal pain
95014/M0.194+T. trichiura eggs (+)Abdominal pain
95027/M0.172+A. lumbricoides, hookworm, S. stercoralis, and T. trichiura eggs (+)Abdominal pain
96023/F0.15+T. trichiura eggs (+)Abdominal pain, vomiting
96035/M0.16+A. lumbricoides and T . trichiura eggs (+)None
10201NA0.258+No informationNo information
10204NA0.438+No informationNo information
10306NA0.164+No informationNo information
10112/F0.021Schistosoma mansoni eggs (+)None
40311/M0.096A. lumbricoides, hookworm, S. stercoralis, and T. trichiura eggs (+)Abdominal pain, constipation
8018/F0.072No helminth eggsNone
80211/M0.056No helminth eggsNone
150212/F0.023No helminth eggsAbdominal pain
220213/M0.001No helminth eggsNone
230312/M0.096S. stercoralis eggs (+)Abdominal pain, vomiting
270111/F0.017A. lumbricoides, hookworm, and T. trichiura eggs (+)None
32046/M0.069A. lumbricoides eggs (+)Abdominal pain
33014/M0.101T. trichiura eggs (+)Abdominal pain
330412/M0.012T. trichiura eggs (+)Abdominal pain
330516/F0.046No helminth eggsNone
35029/F0.052T. trichiura eggs (+)Abdominal pain
39019/F0.005A. lumbricoides, S. stercoralis, and T. trichiura eggs (+)None
400110/F0.032No helminth eggsAbdominal pain
41024/F0.026T. trichiura eggs (+)Abdominal pain, vomiting
490216/M0.031E. vermicularis eggs (+)None
490411/M0.037A. lumbricoides eggs (+)None
50015/F0.027No helminth eggsNone
53016/M0.095A. lumbricoides eggs (+)Diarrhea (sometimes)
630110/F0.087Schistosoma mansoni eggs (+)Abdominal pain
64011/M0.116No helminth eggsAbdominal pain
79018/M0.115A. lumbricoides, hookworm, and T. trichiura eggs (+)Abdominal pain
850311/M0.06A. lumbricoides eggs (+)None
92016/M0.057T. trichiura eggs (+)Diarrhea (sometimes)
970413/F0.124A. lumbricoides and T. trichiura eggs (+)Abdominal pain
1010415/M0.121A. lumbricoides, hookworm, and T. trichiura eggs (+)Abdominal pain
NC0.045
PC**0.48
Cut-off0.135
Figure 1.
Figure 1.

Geographic location of the study area. Jaboatão dos Guararapes is situated in the suburbs of Recife, the capital of the State of Pernambuco. Administratively, the area belongs to Região Metropolitana do Recife.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.103

Figure 2.
Figure 2.

Specificity of the recombinant Toxocara canis antigen. Thirteen children who expelled only Ascaris lumbricoides eggs showed positive reactions against homologous ascarid antigen (A), but none of them reacted with the recombinant T. canis antigen (B). Closed circles indicate serum samples from the 13 children. Each symbols in the panel A corresponds to those in panel B except for the positive controls. Triangles and open circles denote positive and negative controls, respectively. The cut-off value is based on three times the optical density at 415 nm (OD415) value for a pooled negative serum sample.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.103

Figure 3.
Figure 3.

Enzyme-linked immunosorbent assay results using recombinant Toxocara canis antigen. Serum samples examined were divided into six groups based on the absence or presence of helminth eggs. A, No helminth eggs; B, Only Ascaris lumbricoides eggs; C, Only Trichuris trichiura eggs; D, Either hookworm (top two circles), Schistosoma mansoni (bottom and third circle from the bottom), Strongyloides stercoralis (fourth circle from the bottom), or Enterobius vermicularis (second circle from the bottom) eggs; E, Mixed helminth eggs such as A. lumbricoides, T. trichiura, hookworm, and/or Hymenolepis nana; F, individuals without information; G, positive (triangle) and negative controls (open circle) for toxocariasis. The broken line indicates the cut-off value. OD415 = optical density at 415 nm.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.103

Figure 4.
Figure 4.

Dot-blot assay using selected serum samples. In positive cases, the spots appear in the right side upper square. Spots in the left side lower grids are positive controls for secondary antibody. The two panels at the top row (A1 and A2) indicate negative and positive controls for the enzyme-linked immunosorbent assay (ELISA)-positive group. Results from A3 to C8 correspond to code numbers 401–10306 as in Table 1. Panels D1 and D2 are negative and positive controls for ELISA-negative group, and results from D3 to F9 correspond to code numbers 101–10104 in order.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.103

Authors’ addresses: Raquel de Andrade Lima Coêlho and Luiz Bezerra de Carvalho, Jr., Departamento de Bioquímica, Laboratório de Imunopatología Keizo Asami, Universidade Federal de Pernambuco, Campus Universitário, 50670-901 Recife, Pernambuco, Brazil. Emília Pessoa Perez, Departamento de Parasitología, Centro de Pesquisas Aggeu Magalhães-FIOCRUZ, Cidade Universitária, 50670-901 Recife, Pernambuco, Brazil. Kunioki Araki, Section of Environmental Parasitology, Tokyo Medical and Dental University, Graduate School, Tokyo 113-8519, Japan. Tsutomu Takeuchi, Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo 160-8582, Japan. Akira Ito and Hiroshi Yamasaki, Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japan. Takashi Aoki, Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.

Financial support: This work was supported in part by the Japan International Cooperation Agency as a part of an international cooperative project for the Laboratório de Imunopatología Keizo Asami, Universidade Federal de Pernambuco, Brazil.

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Author Notes

Reprint requests: Hiroshi Yamasaki, Department of Parasitology, Asahikawa Medical College, Midorigaoka Higashi 2-1-1-1, Asahikawa 078-8510, Japan. Telephone: 81-166-68-2421. Fax: 81-166-68-2429. E-mail: hyamasak@asahikawa-med.ac.jp.
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