Keywords
toxocariasis - ELISA - IgG - Lorestan
Introduction
Human toxocariasis is a helminthic zoonotic disease caused by the larval stage of Toxocara spp. To date, T. canis and T. cati are identified as contributory agents of human toxocariasis that inhabit the small bowels of dogs and cats as definitive hosts, respectively.[1] Typically, humans may be infected by direct contact with soil contaminated with the feces of dogs with toxocariasis and eating embryonated eggs through contaminated water and food, although ingestion of chicken and cow liver is considered an uncommon route of infection.[2] Although human toxocariasis in immunocompetent individuals is commonly asymptomatic, serious symptoms and complications can occur due to vital organ damage as a result of migrating larvae.[1]
[3] Accordingly, clinical complications of human toxocariasis are identified as visceral larva migrans (VLM), ocular larva migrans (OLM), and neurologic and complex toxocariasis.[1] The identification of human toxocariasis is based on a serological test, enzyme-linked immunosorbent assay (ELISA), by using excretory–secretory antigens from Toxocara larvae. It is extremely hard to determine infective Toxocara larvae in various organs and biopsy samples.[4]
Children in the first and second decades of life are at far more risk of toxocariasis due to contact with the definitive host (especially dogs) and lack of hygiene standards in the consumption of food and water; therefore, it is crucial to study the prevalence of anti-Toxocara antibodies in children in this age range.[5] Based on previous studies, the seroprevalence of infection with toxocariasis in different geographic regions varies from 2 to 90% around the world.[2]
[6] According to the best of our knowledge, there is no study on seroprevalence of human toxocariasis among the children of Lorestan province, western Iran. Therefore, the present study aims to evaluate the seroprevalence and risk factors for T. canis infection in children (2–15 years old) referred to health centers of Lorestan province, Iran.
Materials and Methods
Ethics
This study was approved by Ethics Committee of the Lorestan University of Medical Sciences, Khorramabad, Iran on March 15, 2017. In addition, a written informed consent was obtained from the parents of the children before blood sampling.
Study Design and Sample Collection
This cross-sectional study was performed from August 2016 to March 2017 on 316 children (aged 2–15 years) referred to health centers of Lorestan province, located between the valleys of Zagros Mountains in the west of Iran, bordering the provinces of Markazi, Hamedan, Kermanshah, Khuzestan, Ilam, and Isfahan. Lorestan covers an area of 28,294 km2 with a population of approximately 2 million people. Blood sample (5 mL) was obtained from each patient by venipuncture under sterile conditions. The samples were centrifuged at 1,000 rpm and the sera were stored at −20°C until serological examination.[7]
Questionnaire
Before collection of blood samples, a questionnaire was completed by parents of the children including demographic data (e.g., age, gender), possible risk factors (e.g., animal contacts [dog], and residence), and hypereosinophilia < 10%, one of the main laboratory features of toxocariasis.
Enzyme-Linked Immunosorbent Assay
To measure the anti-T. canis antibodies, serum samples were transported to the Parasitology Laboratory, Department of Parasitology and Mycology, Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences (Khorramabad, Iran) and stored at −20°C until tested. All serum samples were tested using the standard commercially available anti IgG-Toxocara kit (IBL, Germany) according to manufacturer's instructions. The reaction cutoff was calculated as the mean optical density (OD) for negative control sera plus three standard deviations (SDs). The positive and negative control sera present in the kit were included in each plate. The reading was acquired using a microplate reader (BioTek, United States) set at a level of absorbance of 450 nm. All samples were run in triplicate. The results were considered positive when OD450 index was equal to or higher than the cutoff value in ELISA.
Statistical Analysis
In this study, the analytical and descriptive statistics were performed using SPSS 24.0 software (SPSS Inc., Chicago, Illinois, United States). Descriptive statistics were showed in terms of percentage (for categorical) and mean (SD, for continuous) variables. The chi-square test was applied to assess the univariate association between independent variables and outcome. All the variables in univariate analysis (chi-square test) that had a p-value less than 0.25 were entered into multivariate analysis (logistic regression). Multifactorial logistic regression models were used to evaluate association between T. canis seropositivity and the potential risk factors. A p-value < 0.05 was considered to be statistically significant.
Results
Participants
A total of 316 children (aged 2–15 years) referred to the health centers of Lorestan province, Iran were included in the present study. The mean age of the participants was 7.9 ± 2.5 years and most participants were boys (176, 55.7%). In total, 240 participants (75.9%) lived in urban regions, while the remaining (24.1%) lived in rural areas. Among the participants, 85 children (26.9%) were in contact with dogs, whereas 231 children (73.1%) did not have any contact with dogs. Moreover, only 32 children (10.1%) consumed raw or unwashed vegetables and fruits. Among the children, 299 (94.6%) had no hypereosinophilia while 17 (5.4%) had hypereosinophilia >10% ([Table 1]).
Table 1
Demographic characteristics and seroprevalence of anti-T. canis antibodies among children (aged 2–15 years) referred to health centers of Lorestan province, Iran
|
Variables
|
No. (%)
|
IgG positive
|
p-Value
|
|
Gender
|
|
Male
|
176 (55.7)
|
10 (6.25)
|
0.22
|
|
Female
|
140 (44.3)
|
4 (2.85)
|
–
|
|
Age groups
|
|
< 7y
|
105 (33.2)
|
3 (2.9)
|
–
|
|
≥ 7y
|
211 (66.8)
|
11(5.2)
|
0.34
|
|
Residential place
|
|
Urban
|
240 (75.9)
|
5 (2.1)
|
–
|
|
Rural
|
97 (20.1)
|
9 (11.9)[a]
|
<0.001[a]
|
|
Being in contact with dogs
|
|
No
|
231 (73.1)
|
4 (1.73)
|
<0.001[a]
|
|
Yes
|
85 (26.9)
|
10 (11.8)[a]
|
–
|
|
Unwashed vegetables/fruits consumption
|
|
No
|
284 (89.9)
|
13 (4.6)
|
0.70
|
|
Yes
|
32 (10.1)
|
1 (3.1)
|
–
|
|
Hypereosinophilia (>10%)
|
|
No
|
299 (94.6)
|
13 (4.3)
|
–
|
|
Yes
|
17 (5.4)
|
1 (5.9)
|
0.81
|
a
p < 0.05, difference is statistically significant.
Seroprevalence and Risk Factors of T. canis
Of the 316 children, 14 (4.4%) tested seropositive for anti-T.canis IgG antibody. Seroprevalence of anti-T.canis antibody was higher among boys. Ten (5.7%) boys were positive in terms of IgG anti-T. canis antibodies compared with four (2.85%) girls (p = 0.22). Additionally, a chi-square test for trend revealed that the seroprevalence of anti-T. canis IgG antibody did not vary with age among the children between 2 to 15 years (p = 0.34).
Out of the 240 participants living in urban regions, 5 (2.1%) tested seropositive for anti-T. canis antibodies, whereas from 76 participants who lived in rural areas, 9 (11.9%) tested seropositive for anti-T. canis antibodies. There was a significant difference in the prevalence of anti-T. canis antibodies among those living in urban and those living in rural areas (p < 0.001).
Out of 85 children who had contact with dogs, 10 (11.8%) tested seropositive for anti-T. canis antibodies, whereas out of 231 children who had no contact with dogs, 4 (1.73%) tested seropositive for anti-T. canis antibodies. There was a significant difference in the prevalence of anti-T. canis antibodies among children who had contact with dogs and those with no contact with dogs (p < 0.001). Of the 32 children who consumed raw or unwashed vegetables and fruits, 1 (3.1%) was seropositive for anti-T. canis antibodies, while out of 284 children who did not eat raw or unwashed vegetables and fruits, 13 (4.6%) tested seropositive for anti-T. canis antibodies. There was no significant difference in the prevalence of anti-T. canis antibodies among the children who consumed raw or unwashed vegetables and fruits and those who did not eat raw or unwashed vegetables and fruits (p = 0.70).
Out of 299 children who had no hypereosinophilia, 13 (4.3%) children were positive for anti-T. canis antibodies, whereas out of 17 children who had hypereosinophilia > 10%, only 1 child (5.9%) was positive for anti-T. canis antibodies. There was no significant difference in the prevalence of anti-T. canis antibodies among children with no hypereosinophilia and those with hypereosinophilia > 10% (p = 0.83).
[Table 2] shows the association between risk factors and status of anti-T. canis IgG antibodies in the logistic regression analysis. The variables used to evaluate association between risk factors and status of anti-T. canis IgG antibodies were age, gender, area of residence, contact with dogs, and consumption of raw or unwashed vegetables and fruits. Some risk factors that were significantly related to T. canis seropositivity included living in rural regions (p = 0.018) and contact with dogs (p = 0.001). However, other demographic and risk factors did not demonstrate any association with T. canis seropositivity.
Table 2
Logistic regression analysis of potential factors associated with anti-T. canis antibodies among children (aged 2–15 years) referred to health centers of Lorestan province, Iran
|
Variables
|
OR (95% CI)
|
p-Value
|
|
Gender
|
|
Male
|
2.3 (0.7–8.2)
|
0.18
|
|
Female
|
1
|
–
|
|
Residential place
|
|
Urban
|
1
|
–
|
|
Rural
|
4.2 (1.3–13.9)
|
0.018[a]
|
|
Being in contact with dogs
|
|
No
|
1
|
–
|
|
Yes
|
7.9 (2.3–27.3)
|
0.001[a]
|
Abbreviations: CI, confidence interval; OR, odds ratio.
a
p < 0.05 was statistically significant.
Discussion
Toxocariasis as a zoonotic infection is a highly widespread parasitic disease in the tropical and subtropical regions around the world.[7] Humans can generally be infected by ingesting food and water contaminated with embryonated eggs excreted from feces of a definitive host. In humans, the clinical symptoms of disease vary from asymptomatic illness to severe signs, such as ocular, neurological, and/or systemic involvements, a condition known as visceral larva migrans. The diagnosis of human toxocariasis is typically based on serological tests such as ELISA to detect anti-Toxocara antibodies.[1]
[2]
[3] Because treatments are often only partially effective and no safe and effective vaccines exist, it is critical to make efforts to decrease transmission of toxocariasis to reduce the serious manifestations of disease.[5] According to review studies, the seroprevalence rate of toxocariasis varies from 2 to 90% in different geographic regions around the world.[7] Recently, according to a review study conducted by Abdi et al in Iran, the seroprevalence of human toxocariasis, soil contamination with Toxocara spp. eggs, and infections in dogs/cats were reported to be 15.8, 21.6, and 26.8%, respectively.[8] Here, we evaluated the seroprevalence and risk factors for T.canis infection in children (2–15 years old) referred to health centers of Lorestan province, Iran. Out of the 316 children, 14 (4.4%) tested seropositive for anti-T.canis IgG antibody.
The seroprevalence of T. canis infection was less than that reported among children of Jordon,[9] Netherlands,[10] Turkey,[11] Serbia,[12] and Northeast Brazil.[13] In Iran, several studies have been performed on the seroprevalence of T. canis infection among children including the study conducted by Sadjjadi et al;[14] the total prevalence of anti-Toxocara antibodies in the sera of school children of Shiraz, southern Iran was 25.6%. Akhlaghi et al[15] have reported the seroprevalence of anti-Toxocara antibodies to be 8.6% among children (2–15 years old) in Kermanshah province, Iran. In another study, Ghaffar-Naqnehi et al[16] found the seroprevalence of T. canis infection in children 2 to 14 years old referred to health care centers of Chaharmahal and Bakhtiari province, Iran to be only 2%. Moreover, in the study of Alavi et al,[17] the seroprevalence rate of T. canis was 2% in rural and urban school children aged 6 to 15 years in Ahvaz, Iran. Fallah et al[18] found that 29 out of 544 children were positive for anti-Toxocara antibodies showing an overall prevalence of 5.3% in the western Islamic Republic of Iran. These variations in the prevalence of anti-Toxocara antibodies among children in various parts of Iran and the world might be related to sample size, method of testing, transmission routes, and climatic conditions of the studied region and country.[17]
In this study, similar to the work conducted by Sadjjadi et al,[14] we found a significant difference in the prevalence of anti-T. canis antibodies among children living in urban and rural areas (p < 0.01); however, it was in contrast to the findings of Fallah et al[18] and Alaviet al.[17] These variations might be due to work-related activities associated with contact with dogs or having a less hygienic lifestyle. Here, we found that having contact with dogs is a significant risk factor for T. canis seropositivity. Similarly, Ghaffar-Naqnehi et al[16] and Alonso et al[19] demonstrated that contact with dogs is a risk factor for T. canis infection. In the present study, no significant difference was found in age, gender, consumption of raw or unwashed vegetables and fruits, and T. canis seropositivity. In line with these results, Alonso et al[19] and Fallah et al[18] found no significant difference in terms of age, sex, and consumption of unwashed vegetables and fruits. Here, we found that there was no significant difference in the prevalence of anti-T. canis antibodies among children with no hypereosinophilia and those with hypereosinophilia > 10%. In line with our results, some studies in Brazil and Ahvaz also did not demonstrate a significant association between eosinophilia and toxocariasis.[17]
[20]
[21] However, in a study by Alonso et al in Argentina, a significant connection was found between eosinophilia and toxocariasis.[19]
These differences in the seroprevalence of anti-Toxocara antibodies among children could be related to sociocultural behaviors, geographical and environmental parameters, and transmission routes in the populations.[17]
[22]
Conclusion
We found that T. canis infection is prevalent among children (2–15 years old) referred to health centers of Lorestan province, Iran with an overall seroprevalence rate of 4.4%. These findings may be a warning for health centers to pay special attention to toxocariasis among children (2–15 years old) and design screening programs for the prevention of toxocariasis.
